US007.923221B1

(12) United States Patent (10) Patent No.: US 7.923,221 B1 Cabilly et al. (45) Date of Patent: *Apr. 12, 2011

(54) METHODS OF MAKING ANTIBODY HEAVY 4,512.922 A 4, 1985 Jones et al. AND LIGHT CHAINS HAVING SPECIFICITY 4,518,584 A 5, 1985 Mark 4,565,785 A 1/1986 Gilbert et al. FORADESIRED ANTIGEN 4,599,197 A 7, 1986 Wetzel 4,634,665 A 1/1987 Axel et al. (75) Inventors: Shmuel Cabilly, Monrovia, CA (US); 4,642,334 A 2f1987 Moore et al. Herbert L. Heyneker, Burlingame, CA 4,668,629 A 5/1987 Kaplan 4,704,362 A 11/1987 Itakura et al. (US); William E. Holmes, Pacifica, CA 4,713,339 A 12/1987 Levinson et al. (US); Arthur D. Riggs, LaVerne, CA 4,766,075 A 8, 1988 Goeddeletal. (US); Ronald B. Wetzel, San Francisco, 4,792.447 A 12/1988 Uhr et al. CA (US) 4,816,397 A * 3/1989 Boss et al...... 435/68 4,816,567 A 3/1989 Cabilly et al. (73) Assignees: Genentech, Inc, South San Francisco, 4,965,196 A 10, 1990 Levinson et al. 5,081,235 A 1/1992 Shively et al. CA (US); City of Hope, Duarte, CA 5,098,833. A 3/1992 Lasky et al. (US) 5,116,964 A 5/1992 Capon et al. 5,137,721 A 8, 1992 Dallas (*) Notice: Subject to any disclaimer, the term of this 5,149,636 A 9, 1992 Axel et al. patent is extended or adjusted under 35 5,179,017 A 1/1993 Axel et al. 5,225,538 A 7/1993 Capon et al. U.S.C. 154(b) by 0 days. 5,225,539 A 7, 1993 Winter This patent is Subject to a terminal dis 5,336,603 A 8/1994 Capon et al. claimer. 5,420,020 A 5/1995 Riggs 5,428, 130 A 6/1995 Capon et al. 5,455,165 A 10/1995 Capon et al. (21) Appl. No.: 08/422,187 5,500,362 A 3, 1996 Robinson et al. 5,514,582 A 5/1996 Capon et al. (22) Filed: Apr. 13, 1995 5,545.403 A 8/1996 Page 5,545.404 A 8/1996 Page Related U.S. Application Data (Continued) (63) Continuation of application No. 07/205,419, filed on Jun. 10, 1988, now Pat. No. 6,331415, which is a FOREIGN PATENT DOCUMENTS continuation of application No. 06/483,457, filed on AU 194982 2, 1983 Apr. 8, 1983, now Pat. No. 4,816,567. (Continued) (51) Int. C. OTHER PUBLICATIONS CI2N 5/3 (2006.01) Gillis, S.D. & J.S. Wesolowski 1990 Hum. Antibod. Hybridomas CI2N IS/00 (2006.01) 1(1): 47-54.* CI2N 15/63 (2006.01) Owens, R.J. & R.J. Young 1994 Journal of Immunological Methods (52) U.S. Cl...... 435/69.6; 435/252.1; 435/252.3: 168:149-165. 435/252.33:435/254.11: 435/254.21: 435/69.1; Skerra et al 1988 Science 240: 1038-1041. 435/69.7:435/70.1; 435/70.21; 435/71.1; Better et al. 1989 Methods Enzymol 178: 476-496.* 435/320.1; 435/455; 435/483; 435/485; 435/.440; Taylor et al 1988 Mol Cell Biol 8(10): 4197-4203.* 435/.433; 435/438 Letherbarrow 1985 Midec. Immuno (22(4): 407-415.* Field of Classification Search ...... 435/69.6, Raghunathan et al 1996 Prog. Biophy. &Mol. Biol. 65(5): 143.* (58) Wright et al 1991 EMBOJ, 10(10): 2717-2723.* 435/69.1, 325, 252.3: 530/387.3 Buchner et al 1991 Bio/Technology 9:157-162.* See application file for complete search history. Morrison etal Adv. Immunol. (1989).* Horowitz et al. PNAS, (1988).* (56) References Cited Skerrs etal Protein Engineering, (1991).* U.S. PATENT DOCUMENTS (Continued) 4,179,337 A 12, 1979 Davis 4,224.404 A 9, 1980 Viza et al. Primary Examiner — Phillip Gambel 4,237,224 A 12/1980 Cohen (74) Attorney, Agent, or Firm — Sidley Austin LLP 4.338,397 A 7, 1982 Gilbert 4,342,832 A 8, 1982 Goeddeletal. (57) ABSTRACT 4,348,376 A 9/1982 Goldenberg 4,366,246 A 12/1982 Riggs The invention relates to processes for producing an immuno 4,370,417 A 1/1983 Hung globulin or an immunologically functional immunoglobulin 4,399.216 A 8, 1983 Axel et al. 4,403,036 A 9/1983 Hartley fragment containing at least the variable domains of the 4.418,149 A 11, 1983 Ptashine et al. immunoglobulin heavy and light chains. The processes can 4.419,446 A 12/1983 Howley et al. use one or more vectors which produce both the heavy and 4,431,740 A 2f1984 Bell et al. light chains or fragments thereof in a single cell. The inven 4,440,859 A 4, 1984 Rutter et al. 4,444,878 A 4, 1984 Paulus et al. tion also relates to the vectors used to produce the immuno 4.495,280 A 1/1985 Bujard et al. globulin or fragment, and to cells transformed with the vec 4,500,637 A 2/1985 Neville, Jr. et al. tOrS. 4,510,244. A 4, 1985 Parks et al. 4,511,502 A 4, 1985 Builder et al. 47 Claims, 19 Drawing Sheets US 7.923,221 B1 Page 2

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GlaxoWellcome Inc.'s Miscellaneous Motion 11(Suppression of Cabilly Response to Objections to Admissiblity of Evidence. Jan. 16. Certain Deposition Exhibits and Deposition Testimony), Apr. 18. 2001. 2001. Glaxo Response to Cabilly's Objection to Evidence, Jan. 16, 2001. GlaxoWellcome Inc.'s Miscellaneous Motion 12 (Suppression of GlaxoWellcome Miscellaneous Motion 2, Jan. 16, 2001. Deposition Testimony), Apr. 18, 2001. GlaxoWellcome Miscellaneous Motion 3, Jan. 16, 2001. GlaxoWellcome Inc.'s Notice of Change of Real Party in Interest, Order Denying Glaxo Motions Miscellaneous Motions 1 and 2, Jan. Apr. 19, 2001. 29, 2001. Petition from the Apr. 6, 2001 Order of the APJ Under 37 C.F.R. Cabilly Opposition 1 Feb. 2, 2001. 1.644(a)(1), Apr. 20, 2001. Cabilly Opposition 2, Feb. 2, 2001. Memorandum Opinion and Order, Apr. 30, 2001. Cabilly Opposition 3, Feb. 2, 2001. Order Regarding GlaxoWellcome Inc. Motions, May 2, 2001. Cabilly Opposition 4 Feb. 2, 2001. 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Curriculum Vitae of Jack Shively. Motion be the Party Cabilly et al Pursuant to 37 CFRS1.635 to Wagener, Shively publication (Bates Nos. 0927-0934). Replace Exhibits 1-20 Filed on Jan. 8, 1992 With a Corrected Set of Curriculum Vitae of Ron Wetzel. Exhibits and for the Return of Exhibits 1-20 Filed 1-20 on Jan. 8, Wetzel Nbk #1432 (Bates Nos. 0.034-0044, 0.047, 0049-0050, 0053, 1992, Jan. 22, 1992. 0061-0064, 0077-0081, 0087-0088). Corrected Submission of Stipulation Concerning Testimony, Feb. 5, Curriculum Vitae of Jeanne Perry. 1992. Perry Nbk #1290 (Bates Nos. 0.136a-0136b, 0.142-0147, 0149-0154, Cabilly et al. Motion for Extension of Time, Feb. 10, 1992. O156-0158, 0160-0161, 0164-0173, 0181, 0.187-0188, 0.190-0192, Main Brief at Final Hearing of Junior Party Cabilly et al., Feb. 18. 1992. 0 197-0218, 0223, 0237,0244-0261, 0304-0307). Transmittal of Brief for the Party Boss et al., Mar. 18, 1992. Holmes Nbk #1446 (Bates Nos. 0941-0943,0946-0947,0950, 0954 Brief at Final Hearing for Senior Party Boss et al., Mar. 18, 1992. 0957). Cabilly et al. Supplemental Brief at Final Hearing, Apr. 5, 1992. Holmes Spiral #4 (Bates Nos. 0825, 0830,0837-0838, 0840-0843, Reply Brief at Final Hearing of Junior Party Cabilly et al., Apr. 7, 0845-0846,0848-0849, 0852-0853, 0855, 0858). 1992. Holmes Spiral #5 (Bates Nos. 0876, 0881-0882, 0885-0887, 0889). Cabilly etal Motion Pursuant to 37 C.F.R.S 1.635 to Enter Additional Curriculum Vitae of Michael Rey. Pages Into the Cabilly et al Record, Apr. 14, 1992. Rey Nbk #1173 (Bates Nos. 0502, 0504, 0509-0516, 0521-0522, Opposition to Cabilly et al Motion Pursuant to 37 C.F. R. S. 1.635 to 0525-0528,0530-0531,0533-0537, 0541,0543-0544). Enter Additional Pages Into the Cabilly et al Record, Apr. 22, 1992. Curriculum Vitae of Michael Mumford. 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Decision Granting Petition to Correct the Assignee on the Cover Page Transmittal of Preliminary Statement of Boss et al. and Notice to of U.S. Appl. No. 07/205,419, May 7, 2002. Opposing Party, Jun. 4, 1991. Petition Pursuant to 37 C.F.R.S 1.666(b) for Access to Settlement Boss et al. Motion for Benefit of its PCT Application (Boss Motion Agreement (filed by MedImmune), May 8, 2002. 1), Jun. 4, 1991. Order on Petition for Access Pursuant to 35 U.S.C. S165(c) and 37 Boss et al. Motion for Benefit of its British Application (Boss Motion C.F.R.S 1.666(b), Jun. 19, 2002. 2), Jun. 4, 1991. Cabilly et al. Objection to Petition for Access to Settlement Agree Declaration of Timothy John Roy Harris in Support of Boss Motion ment, Jul. 22, 2002. for Benefit of its British Application (Boss Motion 2), Jun. 4, 1991. Celltech's Objection to Petition for Access to Settlement Agreement, Bosset al. Motion for Judgment of Unpatentability of Cabilly Claims Jul. 22, 2002. (Boss Motion 3), Jun. 4, 1991. 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Boss, Interfer rat brain microsomal ribonucleeic acid containing polyadenylate. ence 105,531) pp. 1-4 (Nov. 16, 2005). Wilde C.D. et al. Eur: J. Immunol. 10: 462-267, 1980. Analysis of 48 Federal Register 2696 (LEXSEE 48 FR 2708), effective Feb. 27. immunoglobulin chain secretion using hybrid myelomas. 1983 (Boss Exhibit 1040, Cabilly v. Boss, Interference 105,531) pp. Williamson A.R. Biochem. Soc. Trans. 5: 139-175, 1969. The 1-35 (Jan. 20, 1983). Biosynthesis of multichain proteins. A collection of pictures, graphs and drawings (Cabilly Ex. 2138. Williamson R. et al. Ser: Haemat. IV(3): 23-36, 1971. The isolation Cabilly v. Boss, Interference 105.531). and DNA/RNA hybridization of messenger RNA for globin. A description of experiments and drawings (Cabilly Ex. 2137, Winberry L. et al. J. Immunol. 124(3): 1174-1182, 1980. Cabilly v. Boss, Interference 105.531). Immunoglobulin production and Secretion by variant clones of the A document containing single chain expression, double chain expres MOPC315 mouse myeloma cell line. sion and list of references (Cabilly Ex. 2136, Cabilly v. Boss, Inter Woodland H.R. et al. Developmental Biology 39(1): 134-140, 1974. ference 105,531). The translation of mammalian globin mRNA injected into fertilised Affidavit of Herbert L. Heyneker; EP 0125023 Opposition (Cabilly eggs of Xenopus laevis. Exhibit 2151; Cabilly v. Boss, Interference No. 105,531) (Mar. 20, Yip C.C. et al. Proc, Natl. Acad. Sci. USA 72(12): 4777-4779, 1975. 1997). Translation of messenger ribonucleic acid from isolated pancreatic Affidavit of Ronald Wetzel in U.S. Appl. No. 06/483,457 (Cabilly islets and human insulinomas. Exhibit 2170, Cabilly v. Boss, Interference No. 105,531) (Jul 22, Zehavi-Willner T. et al. Cell 11: 683-693, 1977. Subcellular 1986). compartmentation of albumin and globin made in oocytes under the Alberta et al. Molecular Biology of the Cell, New York:Garland direction of injected messenger RNA. Publishing, Inc. (1983). Herzenberg, L., “Genetic studies with mammalian cells (mice)” Alexander et al., “Y heavy chain disease in man: cDNA sequence Grant ID RO1CA04681 as entered into the CRISP database, Sep. 3, supports partial gene deletion model” Proc. Natl. Acad. Sci. USA 1992 (Abstract only). 79(10):3260-3264 (May 1982). Kabat et al., “Sequences of immunoglobulin chains: tabulation and Allore and Barber, "A recommendation for visualizing disulfide analysis of amino acid sequences of precursors, V-regions, C-regions, bonding by one-dimensional Sodium dodecyl Sulfate— J-chain and beta2-microglobulins . . . .” The Kabat Database of polyacrylamide gel electrophoresis' Analytical Biochemistry Sequences of Proteins of Immunological Interest, 1979, Publication 137(2):523-527 (Mar. 1984). No. 80-2008, p. 185, National Institute of Health, Bethesda, MD. Amzel et al., “The Three Dimensional Structure of a Combining Kuehl, W.M., “Light chain fragments: aberrant expression of Region-Ligand Complex of Immunoglobulin NEW at 3.5-A Reso immunoglobulin genes.” Trends Biochem. Sci., Aug. 1981, pp. 206 lution” Proc. Natl. Acad. Sci. USA 71(4): 1427-1430 (Apr. 1974). 208, vol. 6, No. 8, Elsevier, Cambridge, England. Andersen et al., “Production technologies for monoclonal antibodies Martinis et al., “Monoclonal antibodies with dual antigen specific and their fragments' Curr: Op. Biotechnol. 15:456-462 (2004). ity.” Protides of the Biological Fluids Proceedings Colloquium, vol. Answer and Counterclaim to the Complaint for Declaratory Judg 30, Neuroproteins, Monoclonal Antibodies Separation Methods, ment of Invalidity, Unenforceability, and Noninfringement with 1983, pp. 311-316, vol. 30, Peeters, H. (Ed.), Pergamon Press, Exhibits (GlaxoSmithKline v. Genentech and City of Hope, Case No. Oxford, England. 3:10-cv-00675-JSW) (Mar 10, 2010). 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Cell Press, Cambridge, MA. gene in lymphoid cells using a bovine papillomavirus-1 (BPV-1) Davis, “Immunoglobulin molecules and genes' Microbiology vector” Gene 69(2):349-355 (Sep. 30, 1988). Including Immunology and molecular Genetics, Third edition, 1980, Banerji et al., “A Lymphocyte-specific Cellular Enhancer Is Located Chapter 17, pp. 338-379, Harper & Row, Hagerstown, MD. Downstream of the Joining Region in Immunoglobulin Heavy Chain U.S. Appl. No. 07/233,430, Boss et al. (File History), filed Aug. 18, Genes” Cell 33:729-740 (Jul 1983). 1988. Banerji et al., “Expression of a f-globin gene is enhanced by remote U.S. Appl. No. 07/930,821, Boss et al. (File History), filed Aug. 14. SV40 DNA sequences” Cell 27(2 Pt 1):299-308 (Dec. 1981). 1992. Barrett et al., “Enzyme-linked immunosorbent assay for detection of U.S. Appl. No. 08/165.530, Winter et al. (File History) filed Dec. 13, human antibodies to Salmonellatyphi Viantigen' Journal of Clinical 1993. 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Basic & Clinical Immunology (pp. 28-29, 34-37, 83-95, 254-257. Cabilly List of Exhibits (as filed on Sep. 28, 2007) (Cabilly v. Boss, 266-267, 342-381, 742, 745-746), H. Hugh Fudenberg, 3rd edition, Interference 105,531). Los Altos: Lange Medical Publications (1980). Cabilly Motions List (Cabilly v. Boss, Interference 105.531) (Mar. 2, Bergman and Kuehl, "Co-translational modification of nascent 2007). immunoglobulin heavy and light chains' Journal of Supramolecular Cabilly Notice of Related Proceedings (Cabilly v. Boss, Interference Structure 11(1):9-24 (1979). 105,531) (Jan. 30, 2007). Black's Law Dictionary, Garner, Brian A., 7th edition, St. Paul:West Cabilly Notice of Service of Supplemental Evidence (Responding to Group pp. 129 (1999). Boss's Objection to Cabilly Exhibit 2089) (Cabilly v. Boss, Interfer Bolivar et al., “Construction and Characterization of New Cloning ence 105,531) (Sep. 17, 2007). Vehicles. II. A Multipurpose Cloning System” Gene 2 95-113 Cabilly Opposition 1 (Cabilly v. Boss, Interference 105.531) (Aug. (1977). 24, 2007). Bollen et al., “Expression in Escherichia coli of urokinase antigenic Cabilly Opposition 2 (Opposing Boss Motion to Exclude Evidence) determinants' Biochemical & Biophysical Research Communica (Cabilly v. Boss, Interference 105,531) (Nov. 20, 2007). tions 103(2):391-401 (1981). Cabilly Opposition 3 (Cabilly v. Boss, Interference 105.531) (Oct. Boss List of Exhibits (as updated Oct. 17, 2008) (Cabilly v. Boss, 28, 2008). Interference 105,531). Cabilly Reply (Regarding Boss"Response' to Paper No. 80) (Cabilly Boss List of Exhibits (as updated Sep. 28, 2007) (Cabilly v. Boss, v. Boss, Interference 105,531) (Apr. 8, 2008). Interference 105,531). Cabilly Reply 1 (Reply to Boss Opposition 1 to Cabilly Substantive Boss List of Proposed Motions (Cabilly v. Boss, Interference Motion 1) (Cabilly v. Boss, Interference 105,531) (Sep. 28, 2007). 105,531) (Mar. 2, 2007). Cabilly Reply 2 (Reply to Boss Opposition 2 to Cabilly Substantive Boss Miscellaneous Motion 2 (Motion to Exclude Evidence) Motion 2) (Cabilly v. Boss, Interference 105,531) (Sep. 28, 2007). (Cabilly v. Boss, Interference 105,531) (Oct. 31, 2007). Cabilly Reply 4 (Reply to Boss Opposition 4) (Cabilly v. Boss, Boss Miscellaneous Motion 3 (Motion to Strike Cabilly Reply 5) Interference 105,531) (Oct. 10, 2008). (Cabilly v. Boss, Interference 105,531) (Oct. 21, 2008). Cabilly Reply 5 (Reply to Boss Opposition 5) (Cabilly v. Boss, Boss Notification of Notice of Appeal (Cabilly v. Boss, Interference Interference 105,531) (Oct. 10, 2008). 105,531) (Feb. 11, 2009). Cabilly Response to Paper No. 80 (Cabilly v. Boss, Interference Boss Objection to Served Evidence Served with Cabilly Reply 1 105,531) (Mar. 17, 2008). (Cabilly v. Boss, Interference 105,531) (Oct. 5, 2007). Cabilly Substantive Motion 1 (For Judgment Based on Estoppel) Boss Opposition 1 (Cabilly v. Boss, Interference 105.531) (Aug. 24. (Cabilly v. Boss, Interference 105,531) (May 25, 2007). 2007). Cabilly Substantive Motion 2 (For Judgment under 35 U.S.C. 102(g)) Boss Opposition 2 (Cabilly v. Boss, Interference 105.531) (Aug. 24. (Cabilly v. Boss, Interference 105,531) (May 25, 2007). 2007). Cabilly Substantive Motion 4 (To Change Benefit Accorded Boss) Boss Opposition 4 (Cabilly v. Boss, Interference 105.531) (Aug. 29, (Cabilly v. Boss, Interference 105,531) (Jun. 27, 2008). 2008). Cabilly Substantive Motion 5 (For Judgement on Priority) (Cabilly v. Boss Opposition 5 (Cabilly v. Boss, Interference 105.531) (Aug. 29. Boss, Interference 105,531) (Jun. 27, 2008). 2008). Cabilly Supplemental Notice of Related Proceedings (Cabilly v. Boss Reply 1 (Cabilly v. Boss, Interference 105,531) (Sep. 28, 2007). Boss, Interference 105,531) (Sep. 17, 2007). Boss Reply 2 (Motion to Exclude Evidence) (Cabilly v. Boss, Inter Cabilly Tutorial (Cabilly v. Boss, Interference 105,531) (May 25, ference 105.531) (Dec. 5, 2007). 2007). Boss Reply 3 (Cabilly v. Boss, Interference 105,531) (Nov. 4, 2008). Cabilly's Response to Request for Prior Art (in responding to the Boss Response to Cabilly Reply 4–Additional Material Facts memorandum opinion and order (paper No. 3) dated Jan. 16, 2007 (Cabilly v. Boss, Interference 105,531) (Oct. 17, 2008). (Cabilly v. Boss, Interference 105,531) (Mar. 2, 2007). Boss Response to Cabilly Response to Paper No. 80 (Cabilly v. Boss, Cabilly's Responsive Paper Discussing Moore Patent (in responding Interference 105,531) (Apr. 4, 2008). to the memorandum opinion and order (paper No. 3) dated Jan. 16. Boss Response to Memorandum Opinion and Order (Cabilly v. Boss, 2007 (Cabilly v. Boss, Interference 105,531) (Mar. 2, 2007). Interference 105,531) (Mar. 2, 2007). Cabilly's Updated List of Exhibits (as of Nov. 12, 2008) (Cabilly v. Boss Substantive Motion 1 (for judgment based on obviousness-type Boss, Interference 105,531) (Nov. 12, 2008). double patenting) (Cabilly v. Boss, Interference 105,531) (May 25, Calendar for year 1983 (Cabilly Exhibit 2153, Cabilly v. Boss, Inter 2007). ference No. 105,531) (1983). Boss UK application GB 8308235 (Cabilly Ex. 2186, Cabilly v. Boss, Celltech R&D Ltd.'s Reply Brief in Support of Its Motion for Judg Interference 105,531) (Mar. 25, 1983). ment on the Pleadings (Redacted, Non-Confidential Version), Boss' Response to Cabilly Reply 5—Additional Material Facts Medimmune v. Genentech, City of Hope, and Celltech, Case No. CV (Cabilly v. Boss, Interference 105,531) (Oct. 17, 2008). 03-02567 MRP (CTx) (Cabilly Ex. 2127, Cabilly v. Boss, Interfer Boven and Pinedo, "Monoclonal antibodies in cancer treatment: ence 105,531) (Dec. 5, 2003). where do we stand after 10 years?” Radiotherapy & Oncology 5 Centocor's Reply to Defendants' First Amended Counterclaims (2):109-117 (Feb. 1986). (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 Bowden et al., "Cloning of eukaryotic genes in single-strand phage MRP (CTx)) (Nov. 26, 2008). vectors: the human interferon genes' Gene 27(1):87-99 (Jan. 1984). Centocor, Inc.'s Opening Briefon Claim Construction with Exhibits Braun et al., “The second century of the antibody. Molecular per (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 spectives in regulation, pathophysiology, and therapeutic applica MRP (CTx)) (Apr. 14, 2009). tions' Western Journal of Medicine 157(2): 158-168 (Aug. 1992). Chang et al., “Phenotypic Expression in E. coli of a DNA Sequence Brekke and Sandlie, "Therapeutic antibodies for human diseases at Coding for Mouse Dihydrofolate Reductase” Nature 275:617-624 the dawn of the twenty-first century” Nature Reviews. Drug Discov (Oct. 19, 1978). ery 2(1):52-62 (Jan. 2003). City of Hope's Answer to First Amended Complaint and Affirmative Brousseau et al., “Synthesis of a human insulin gene V. Enzymatic Defenses, Jury Trial Demanded (Centocor v. Genentech and City of assembly, cloning and characterization of the human proinsulin Hope, Case No. CV 08-03573 MRP (CTx)) (Sep. 19, 2008). DNA” Gene 17:279-289 (1982). City of Hope's Answer to Second Amended Complaint and Affirma Burton, D., "Human Monoclonal Antibodies: Achievement and tive Defenses, Jury Trial Demanded (Centocor v. Genentech and City Potential” Hospital Practice 27(8):67-74 (Aug. 15, 1992). of Hope, Case No. CV 08-03573 MRP (CTx)) (Jul 14, 2009). Cabilly Exhibit List (Interference 104,532). Civil MInutes—General, Scheduling Conference (Centocor v. Cabilly List of Exhibits (as filed on Sep. 17, 2007) (Cabilly v. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) BossInterference 105,531). (Feb. 9, 2009). US 7.923,221 B1 Page 22

Claim Construction Order (Centocor v. Genentech and City of Hope, Declaration for Patent Application dated Apr. 4, 1983 and Apr. 5, Case No. CV 08-03573 MRP (CTx)) (Jun. 8, 2009). 1983 (Cabilly Exhibit 2140, Cabilly v. Boss, Interference 105,531) Claim Construction Order (MedImmune v. Genentech, Case No. CV (1983). 03-2567 MRP (CTx)) (Aug. 16, 2007). Declaration of Arthur D. Riggs, Ph.D., filed on Jun. 27, 2008 (Cabilly Clarification (Decision on Cabilly request for clarification) (Cabilly Exhibit 2200, Cabilly v. Boss, Interference No. 105,531) (Jun. 20, v. Boss, Interference 105,531) (Dec. 30, 2008). 2008). Cohen et al., “Nonchromosomal Antibiotic Resistance in Bacteria: Declaration of Arthur Riggs in Patent Interference 102,572 (Cabilly Genetic Transformation of Escherichia coli by R-Factor DNA” Proc. Exhibit 2144, Cabilly v. Boss, Interference 105,531) (Oct. 28, 1991). Natl. Acad. Sci. USA 69(8): 2110-2114 (Aug. 1972). Declaration of Daralyn J. Durie in Support of Defendants Genentech, Comparison of Draft U.S. Appl. No. 06/483,457 dated Mar. 25, 1983 Inc. and City of Hope's Motion to Transfer with Exhibits with U.S. Appl. No. 06/483,457, filed Apr. 8, 1983 (Cabilly Ex. 2124. (GlaxoSmithKline v. Genentech and City of Hope, Case No. 3:10-cv Cabilly v. Boss, Interference 105.531). 00675-JSW) (Mar. 10, 2010). Comparison of Draft U.S. Appl. No. 06/483,457 dated Mar. 31, 1983 Declaration of Dennis R. Burton, Ph.D. with Exhibit (Boss Exhibit with U.S. Appl. No. 06/483,457, filed Apr. 8, 1983 (Cabilly Ex. 2125, 1004, Cabilly v. Boss, Interference 105,531) (May 25, 2007). Cabilly v. Boss, Interference 105.531). Declaration of Dr. Kate H. Murashige, Esq., filed Jun. 27, 2008 Comparison of Draft Application of U.S. Appl. No. 06/483,457 dated (Cabilly Exhibit 2195, Cabilly v. Boss, Interference No. 105.531) Feb. 25, 1983 and U.S. Appl. No. 06/483:457, filed Apr. 8, 1983 (Jun. 23, 2008). (Cabilly Ex. 2123, Cabilly v. Boss, Interference 105,531). Declaration of Dr. Mary-Jane Gething with Exhibit (Centocor v. Complaint for Declaratory Judgment (Centocor v. Genentech & City Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) of Hope, case No. CV08-03573PA (AGRx), C.D. Cal.) (May 30, (Mar. 24, 2009). 2008). Declaration of Dr. Richard Axel, filed Oct. 5, 1999 in US Appl. No. Complaint for Declaratory Judgment of Invalidity, Unenforceability, 08/422, 187 with Exhibits. and Noninfringement with Exhibits (GlaxoSmithKline v. Genentech Declaration of Geoffrey Thomas Yarranton (Received at PTO on Jul. and City of Hope, Case No. 3:10-cv-00675-JSW) (Feb. 17, 2010). 17, 1995 for U.S. Appl. No. 08/450,727, Cabilly Ex. 2078A, Cabilly Complaint with Exhibits (GlaxoSmithKline v. Genentech and City of v. Boss, Interference 105,531) (Jun. 1, 1995). Hope, Civil Action 09-61608) (Oct. 8, 2009). Declaration of Geoffrey Thomas Yarranton, filed Apr. 3, 1996 in U.S. Cordingley and Preston, “Transcription and translation of the herpes Appl. No. 08/165.530 (Cabilly Ex. 2094, Cabilly v. Boss, Interfer simplex virus type 1 thymidine kinase gene after microinjection into ence 105,531) (Mar. 13, 1996). Declaration of Geoffrey Thomas Yarranton, filed in U.S. Appl. No. Xenopus laevis oocytes' Journal of General Virology 54 (Pt 2):409 08/233,430 (Cabilly Ex. 2070, Cabilly v. Boss, Interference 105.531) 414 (Jun. 1981). (Mar. 24, 1992). Correspondence between counsel for Cabilly and counsel for Boss Declaration of Geoffrey Thomas Yarranton, filed in U.S. Appl. No. regarding joint response to Paper No. 80 in Interference 105,531 08/450,727 (Cabilly Ex. 2078, Cabilly v. Boss, Interference 105.531) (Cabilly Ex. 2126, Cabilly v. Boss, Interference 105,531) (Mar. (Jun. 1, 1995). 2008). Declaration of Herbert L. Heyneker, Ph.D., filed Jun. 27, 2008 Cosimi et al., “Use of monoclonal antibodies to T-cell Subsets for (Cabilly Exhibit 2198. Cabilly v. Boss, Interference No. 105.531) immunologic monitoring and treatment in recipients of renal (Jun. 24, 2008). allografts' New England J. of Medicine 305(6):308-314 (Aug. 6. Declaration of Ian A. Wilson, D. Phil., D.S.C., F.R.S. (Cabilly Ex. 1981). 2066, Cabilly v. Boss, Interference 105.531) (May 22, 2007). Cover letter, Extension of Time, Reply, and Terminal Disclaimer in Declaration of Jeanne Perry in Patent Interference No. 102.572 U.S. Appl. No. 08/422, 187 (Cabilly Exhibit 2201, Cabilly v. Boss, (Cabilly Exhibit 2179, Cabilly v. Boss, Interference No. 105.531) Interference No. 105,531) (Mar. 3, 2003). (Oct. 27, 1991). Crea et al., “Chemical Synthesis of Genes for Human Insulin' Proc Declaration of L. Jeanne Perry, Ph.D., filed Jun. 27, 2008 (Cabilly Natl AcadSci US A75(12):5765-5769 (Dec. 1978). Exhibit 2203, Cabilly v. Boss, Interference No. 105,531) (Jun. 23, Croce et al., “Chromosomal location of the genes for human 2008). immunoglobulin heavy chains” Proc. Natl. Acad. Sci. USA 76 Declaration of Marcus E. Sernel in Support of Genentech, Inc.'s and (7):3416-3419 (Jul 1979). City of Hope's Opening Brief on Claim Construction with Exhibits Croce et al., “Preferential retention of human chromosome 14 in (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 mouse X human B cell hybrids' European Journal of Immunology MRP (CTx)) (Mar. 24, 2009). 10:486-488 (1980). Declaration of Mark Lemley in Support of Defendants Genentech, Cross-Examination of Michael Botchan, Ph.D. (Boss Exhibit 1043, Inc. and City of Hope's Motion to Transfer (GlaxoSmithKline v. Cabilly v. Boss, Interference 105,531) (Jul. 31, 2008). Genentech and City of Hope, Case No. 3:10-cv-00675-JSW) (Mar. Cross-Examination of Ronald B. Wetzel, Ph.D. (Boss Exhibit 1039 10, 2010). Cabilly v. Boss, Interference 105.531) (Aug. 5, 2008). Declaration of Martin P. Hoffman, Esquire, filed Jun. 27, 2008 Cross-Examination of Shmuel Cabilly (Boss Exhibit 1042, Cabilly v. (Cabilly Exhibit 2190, Cabilly v. Boss, Interference No. 105.531) Boss, Interference 105,531) (Aug. 12, 2008). (Jun. 24, 2008). Curriculum Vitae of Ian Andrew Wilson, D. Phil., D.Sc., F.R.S. Declaration of Michael Botchan under 37 CFR 1.132 with Exhibit, (Cabilly Exhibit 2067, Cabilly v. Boss, Interference 105,531). filed in Reexamination Control Nos. 90/007,542 and 90/007,859 Curriculum Vitae of Steven Lanier McKnight (Cabilly Exhibit 2107. (Cabilly Exhibit 2193, Cabilly v. Boss, Interference No. 105.531) Cabilly v. Boss, Interference 105.531). (May 20, 2007). Dalla-Favera et al., “Human c-myc onc gene is located on the region Declaration of Michael Botchan, Ph.D., filed Jun 27, 2008 (Cabilly of chromosome 8 that is translocated in Burkitt lymphoma cells' Exhibit 2187. Cabilly v. Boss, Interference No. 105,531) (Jun. 23, Proc. Natl. Acad. Sci. USA 79(24):7824-7827 (Dec. 1982). 2008). Date stamped postcard from PTO (Cabilly Exhibit 2158, Cabilly v. Declaration of Ronald B. Wetzel, Ph.D., filed Jun. 27, 2008 (Cabilly Boss, Interference No. 105,531) (Apr. 8, 1983). Exhibit 2199, Cabilly v. Boss, Interference No. 105,531) (Jun. 24, Date stamped receipt from PTO dated Apr. 8, 1983; cover letter dated 2008). Apr. 22, 1983 from De Gastyne to Johnston; and debit note dated Apr. Declaration of Ronald Wetzel in Patent Interference No. 102,572 22, 1983 (Cabilly Exhibit 2143, Cabilly v. Boss, Interference (Cabilly Exhibit 2169, Cabilly v. Boss, Interference No. 105.531) 105.531) (Apr. 1983). (Oct. 28, 1991). de Saint Vincent et al., “The Cloning and Reintroduction into Animal Declaration of Shmuel Cabilly in Patent Interference No. 102.572 Cells of a Functional CAD Gene, a Dominant Amplifiable Genetic (Cabilly Exhibit 2162, Cabilly v. Boss, Interference No. 105.531) Marker Cell 27(Part 1):267-277 (Dec. 1981). (Oct. 28, 1991). US 7.923,221 B1 Page 23

Declaration of Shmuel Cabilly, Ph.D. (In Support of Priority of Deposition of William E. Holmes, Ph.D. (Centocor v. Genentech and Invention), filed Jun. 27, 2008 (Cabilly Exhibit 2204, Cabilly v. Boss, City of Hope, Case No. CV 08-03573 MRP (CTx)) (Apr. 30, 2010). Interference No. 105,531) (Jun. 25, 2008). Deposition Transcript of Arthur D. Riggs, M.D. with Exhibits Declaration of Steven Lanier McKnight, filed Aug. 24, 2007 (Cabilly (Medimmune, Inc. v. Genentech, Inc. and City of Hope, Case No. Ex. 2093, Cabilly v. Boss, Interference 105,531) (Aug. 23, 2007). CV03-2567 MRP (CTx)) (Nov. 16, 2007). Declaration of William E. Holmes, Ph. D., filed Jun. 27, 2008 Deposition Transcript of Danny Huntington, Esq. (Medimmune, Inc. (Cabilly Exhibit 2188. Cabilly v. Boss, Interference No. 105.531) v. Genentech, Inc and City of HopeCase No. CV03-2567 MRP (Jun. 20, 2008). (CTx)), (Dec. 18, 2007). Declaration of William Holmes in Patent Interference No. 102,572 Deposition Transcript of E. Fintan Walton, Ph.D. (Medimmune v. (Cabilly Exhibit 2159, Cabilly v. Boss, Interference No. 105.531) Genentech and City of Hope, Case No. 03-2567 MRP (CTx)) (Apr. (Oct. 28, 1991). 23, 2008). Declaration of Yvonne Bobadilla, filed Jun. 27, 2008 (Cabilly Exhibit Deposition Transcript of Ginger Dreger with Exhibits (Medimmune, 2202, Cabilly v. Boss, Interference No. 105,531) (Jun. 24, 2008). Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP Defendant Genentech, Inc.'s and City of Hope's Counterclaims, Jury (CTx)) (Oct. 17, 2007). Trial Demanded with Exhibits (Centocor v. Genentech and City of Deposition Transcript of Henry Lowman, Ph.D. (Medimmune, Inc. v. Hope, Case No. CV 08-03573 MRP (CTx)) (Sep. 19, 2008). Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) Defendant Genentech, Inc.'s and City of Hope's First Amended (Feb. 6, 2008). Counterclaims, Jury Trial Demanded with Exhibits (Centocor v. Deposition Transcript of Herbert Heyneker with Exhibits (Medim Gerientech and City of Hope, Case No. CV 08-03573 MRP (CTx)) mune, Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 (Oct. 10, 2008). MRP (CTx)) (Oct. 28, 2007). Defendant Genentech, Inc.'s Answer to First Amended Complaint Deposition Transcript of Ian M. Armitage with Exhibits (Medim and Affirmative Defenses, Jury Trial Demanded (Centocor v. mune, Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) MRP (CTx)) (Oct. 16, 2007). (Sep. 19, 2008). Deposition Transcript of Jeanne Perry, Ph.D. (Medimmune, Inc. v. Defendant Genentech, Inc.'s Answer to Second Amended Complaint Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) and Affirmative Defenses, Jury Trial Demanded (Centocor v. (Jan. 18, 2008). Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Deposition Transcript of Laurie H. Glimcher, M.D. (Medimmune, (Jul 14, 2009). Defendants Genentech Inc.'s and City of Hope's Second Amended Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP Counterclaims, Jury Trial Demand (Centocor v. Genentech and City (CTx)) (Apr. 22, 2008). of Hope, Case No. CV 08-03573 MRP (CTx)) (Jul. 1, 2009). Deposition Transcript of Matthew P. Scott, Ph.D. (Medimmune, Inc. Defendants Genentech, Inc. and City of Hope's Motion to Transfer; v. Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP Memorandum of Points and Authorities (GlaxoSmithKline v. (CTx)) (Apr. 16, 2008). Genentech and City of Hope, Case No. 3:10-cv-00675-JSW) (Mar. Deposition Transcript of Max D. Hensley with Exhibits (Medim 10, 2010). mune, Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 Defendants' Motion to Dismiss or in the Alternative Transfer Action MRP (CTx)) (Oct. 24, 2007). to the United States District Court for the Central District of Califor Deposition Transcript of Ronald Burnell Wetzel with Exhibits nia and Supporting Memorandum of Law (GlaxoSmithKline v. (Medimmune, Inc. v. Genentech, Inc. and City of Hope, Case No. Genentech and City of Hope, Civil Action 09-61608) (Dec 16, 2009). CV03-2567 MRP (CTx)) (Nov. 29, 2007). Deposition of Arthur D. Riggs, Ph.D. with Exhibits (Centocor v. Deposition Transcript of Scott Chambers, Ph.D. (Medimmune, Inc. v. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) (Mar. 26, 2010). (Apr. 24, 2008). Deposition of Dennis Burton, Ph.D. (Boss Exhibit 1035, Cabilly v. Deposition Transcript of Sean Johnston (Medimmune, Inc. v. Boss, Interference 105,531) (Aug. 2, 2007). Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) Deposition of Dennis Burton, Ph.D. (Boss Exhibit 1051, Cabilly v. (Nov. 9, 2007). Boss, Interference 105,531) (Sep. 24, 2008). Deposition Transcript of Sharon E. Crane, Ph.D. (Medimmune, Inc. v. Deposition of Dennis R. Burton, Ph.D. (Cabilly Ex. 2092, Cabilly v. Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) Boss, Interference 105,531) (Aug. 2, 2007). (Dec. 18, 2007). Deposition of Herbert Heyneker, Ph.D. with Exhibits (Centocor v. Deposition Transcript of Shmuel Cabilly (with exhibits, Medim Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) mune, Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 (Apr. 2, 2010). MRP (CTx)) (Nov. 1, 2007). Deposition of John E. Shively with Exhibits (Centocor v. Genentech Deposition Transcript of Timothy R. Schwartz (Medimmune, Inc. v. and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Mar. 2, Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP (CTx)) 2010). (Dec. 4, 2007). Deposition of John McLaughlin (Centocor v. Genentech and City of Deposition Transcript of William E. Holmes with Exhibit (Medim Hope, Case No. CV 08-03573 MRP (CTx)) (Jul. 31, 2009). mune, Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 Deposition of Kate H. Murashige with Exhibits (Centocor v. MRP (CTx)) (Oct. 19, 2007). Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Diagram (Plaintiff Exhibit 76, Centocor v. Genentech and City of (Mar. 9, 2010). Hope, Case No. CV 08-03573 MRP (CTx)). Deposition of Kate H. Murashige, Ph.D. (Boss Exhibit 1041, Cabilly Diagram (Plaintiff Exhibit 77, Centocor v. Genentech and City of v. Boss, Interference 105.531) (Aug. 7, 2008). Hope, Case No. CV 08-03573 MRP (CTx)). Deposition of L. Jeanne Perry with Exhibits (Centocor v. Genentech Dillman et al., “Therapy of chronic lymphocytic leukemia and cuta and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Apr. 30, neous T-cell lymphoma with T101 monoclonal antibody” Journal of 2010). Clinical Oncology 2(8):881-891 (Aug. 1984). Deposition of Ronald Wetzel with Exhibits (Centocor v. Genentech Dillman, R., "Monoclonal antibodies in the treatment of cancer' and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Jan. 28, Critical Reviews in Oncology-Hematology 1 (4):357-385 (1984). 2010). Docket report as of Sep. 17, 2007, Medimmune Inc. v. Genentech, Deposition of Shmuel Cabilly, Ph.D. with Exhibits (Centocor v. Inc., U.S. District Court, Central District Court of California, Case Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) No. 2:03-cv-02567-MRP-CT (Cabilly Ex. 2112, Cabilly v. Boss, (Apr. 9, 2010). Interference 105,531). Deposition of Wendy M. Lee (Centocor v. Genentech and City of Document entitled “Legend to Figures” (Cabilly Exhibit 2165, Hope, Case No. CV 08-03573 MRP (CTx)) (Apr. 27, 2010). Cabilly v. Boss, Interference No. 105.531). US 7.923,221 B1 Page 24

Draft Application of U.S. Appl. No. 06/483,457 (Annotated with Expert Report of Matthew P. Scott, Ph.D. with Exhibits (MedIm handwritten line numbers, Cabilly Ex. 2120A, Cabilly v. Boss, Inter mune, Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 ference 105.531) (Feb. 25, 1983). MRP (CTx), C.D.Cal.)) (Mar. 1, 2008). Draft Application of U.S. Appl. No. 06/483,457 (Cabilly Ex. 2120, Expert Report of Michel C. Nussenzweig, M.D., Ph.D. with Exhibits Cabilly v. Boss, Interference 105,531) (Feb. 25, 1983). (MedImmune, Inc. v. Genentech, Inc. and City of Hope (Case No. Draft Application of U.S. Appl. No. 06/483,457 (Cabilly Ex. 2121, CV03-2567 MRP (CTx), C.D.Cal.)) (Jan. 25, 2008). Cabilly v. Boss, Interference 105,531) (Mar. 25, 1983). Expert Report of Randolph Wall, Ph.D. (Centocor v. Genentech and Draft Application of U.S. Appl. No. 06/483,457 (Cabilly Ex. 2122, City of Hope, Case No. CV 08-03573 MRP (CTx)) (May 14, 2010). Cabilly v. Boss, Interference 105,531) (Mar. 31, 1983). Expert Report of Robert B. Freedman with Exhibits (MedImmune, Dunnick et al., “A mouse immunoglobulin heavy chain deletion Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP (CTx), C.D.Cal.)) (Feb. 29, 2008). mutant: isolation of a cDNA clone and sequence analysis of the Expert Reports of Dennis R. Burton, Ph.D. And Jonathan S. Weiss mRNA' Nucleic Acids Research 8(7): 1475-1484 (Apr. 11, 1980). man, Ph.D., Genentech, Inc. v. Celltech Therapeutics, Ltd., Case No. Ebersman, D., “Investment Community Meeting: Financial Over C98-3926 MMC ENE (Cabilly Ex. 2089A, Cabilly v. Boss, Interfer view” (Slides presented on Genentech, Inc. webcast) pp. 1-23 (Mar. ence 105,531) (Apr. 14, 2000). 14, 2008). Expert Witness Report of Scott A.M. Chambers, Ph.D. with Exhibits Echols and Murialdo, “Genetic map of bacteriophage lambda” (MedImmune, Inc. v. Genentech, Inc. and City of Hope (Case No. Microbiological Reviews 42(3):577-591 (Sep. 1978). CV03-2567 MRP (CTx), C.D.Cal.)) (Jan. 24, 2008). Edelman et al., “Reconstitution of Immunologic Activity by Interac Express Mail Post Office Receipt dated Mar. 22, 1983 and City of tion of Polypepitcle Chains of Antibodies' Proc. Natl. Acad. Sci. USA Hope Request for Petty Cash (Cabilly Exhibit 2166, Cabilly v. Boss, 50:753-761 (1963). Interference No. 105.531) Mar. 22, 1983). Edelman et al., “The covalent structure of an entire YG Express mail receipt dated Mar. 29, 1983 and City of Hope Request immunoglobulin molecule” Proc. Natl. Acad. Sci. USA 63 78-85 for Petty Cash (Cabilly Ex. 2139, Cabilly v. Boss, Interference (1969). 105,531) (1983). Edelman, "Antibody Structure and Molecular Immunology” (Nobel First Amended Complaint for Declaratory Judgment (Centocor v. Lecture: Physiology or Medicine) pp. 31-54 (Dec. 12, 1972). Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Edge et al., “Total Synthesis of a Human Leukocyte Interferon Gene” (Sep. 3, 2008). 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Boss, Inter cultured mammalian cells: evidence for homologous recombination ference 105.531) (Mar. 9, 2007). between injected plasmid DNA molecules' Molecular & Cellular Etkin and Maxson Jr., “The synthesis of authentic sea urchin tran Biology 2(11): 1372-1387 (Nov. 1982). Scriptional and translational products by sea urchin histone genes Foon et al., “Effects of monoclonal antibody therapy in patients with injected into Xenopus laevis oocytes' Developmental Biology 75(1) chronic lymphocytic leukemia' Blood 64(5) : 1085-1093 (Nov. :13-25 (Mar. 1980). 1984). Excerpts from Prosecution File History (Exhibit 1173, Glaxo v. Freedman and Sela, "Recovery of specific activity upon reoxidation Cabilly, Interference No. 104,532, dated 1994-1995). of completely reduced polyalanyl rabbit antibody” Journal of Bio Excerpts from the prosecution history of U.S. Appl. No. 08/165.530 logical Chemistry 241 (22):5225-5232 (Nov. 25, 1966). (Winter et al.) filed Dec. 13, 1993 (dated 1996-1997). Genentech, Inc.'s and City of Hope's Corrected Opening Brief on Expense Reimbursement Document (Cabilly Exhibit 2150; Cabilly Claim Construction (Centocor v. Genentech and City of Hope, Case v. Boss, Interference No. 105.531). No. CV 08-03573 MRP (CTx)) (Apr. 2, 2009). Expert Rebuttal Report of John E. Shively, Ph.D. (Plaintiff Exhibit Genentech, Inc.'s and City of Hope's Opening Brief on Claim Con 134, Centocor v. Genentech and City of Hope, Case No. CV struction (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) (May 5, 2000). 08-03573 MRP (CTx)) (Mar. 24, 2009). Expert Report of Arne Skerra, Ph.D. (Centocor v. Genentech and City Genentech, Inc.'s and City of Hope's Reply Brief on Claim Con of Hope, Case No. CV 08-03573 MRP (CTx)) (May 14, 2010). struction (Centocor v. Genentech and City of Hope, Case No. CV Expert Report of Carlo M. Croce, M.D. with Exhibits (MedImmune, 08-03573 MRP (CTx)) (Apr. 28, 2009). Inc. v. Genentech, Inc. and City of Hope, Case No. CV03-2567 MRP Genentech, Inc., “Genentech and Lilly Reach Settlement” (Press (CTx), C.D.Cal.) (Mar. 1, 2008). release: Boss Exhibit 1030, Cabilly v. Boss, Interference 105.531) pp. Expert Report of E. Fintan Walton (Medimmune v. Genentech and 1 (Jan. 5, 1995). City of Hope, Case No. CV03-2567 (CTx)) (Feb. 29, 2008). Gerhard et al., “Repertoire of antiviral antibodies expressed by Expert Report of Esther M. Kepplinger with Exhibits (MedImmune, somatic cell hybrids” Proc. Natl. Acad. Sci. USA 75(3):1510-1514 Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP (Mar. 1978). (CTx), C.D.Cal.)) (Mar. 1, 2008). Gheysen and Fiers, “Expression and excretion of human fibroblast B Expert Report of Eugene RZucidlo (Centocor v. Genentech and City interferon in monkey cells after transfection with a recombinant of Hope, Case No. CV 08-03573 MRP (CTx)) (May 14, 2010). SV40 plasmid vector” Journal of Molecular & Applied Genetics 1 Expert Report of James A. Forstner with Exhibits (MedImmune, Inc. (5):385-394 (1982). v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP Glaxo Group Limited and GlaxoSmithKline's Notice of Dismissal (CTx), C.D.Cal.)) (Jan. 24, 2008). Without Prejudice (GlaxoSmithKline v. Genentech and City of Hope, Expert Report of Kathryn L. Calame, Ph.D. with Exhibits (MedIm Civil Action 09-61608) (Feb. 17, 2010). mune, Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 GlaxoWellcome Inc. Exhibit List (Glaxo v. Cabilly, Interference MRP (CTx), C.D.Cal.)) (Jan. 25, 2008). 104,532). Expert Report of Laurie H. Glimcher, M.D. with Exhibits (MedIm Goeddel et al., “Expression in Escherichia coli of chemically syn mune, Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 thesized genes for human insulin' Proc. Natl. Acad. Sci. USA 76 MRP (CTx), C.D.Cal.)) (Jan. 25, 2008). (1):106-110 (1979). 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Lennox, E., "Monoclonal antibodies and tumor antigens—a perspec Miller et al., “Considerations for treatment with hybridoma antibod tive' Hybridomas in Cancer Diagnosis and Treatment (Progress in ies' Hybridomas in cancer diagnosis and treatment (Progress in Cancer Research and Therapy vol. 21), Mitchell et al., New York: Cancer Research and Therapy, vol. 21), Mitchell et al., New York: Raven Press pp. 5-13 (1982). Raven Press pp. 133-145 (1982). Letter from Ronald Wetzel to George Rose regarding IgG (Plaintiff Miller et al., “Treatment of B-cell lymphoma with monoclonal anti Exhibit 78, Centocor v. Genentech and City of Hope, Case No. CV idiotype antibody” New England J. of Medicine 306 (9):517-522 08-03573 MRP (CTx)) (Apr. 14, 1983). (Mar. 4, 1982). Letter to Martin P. Hoffman from Linda K. Johnston (Cabilly Ex. Milstein et al., “Interchain Disulphide Bridges of Mouse Immunoglobulin M” Biochemical Journal 151:615-624 (1975). 2141, Cabilly v. Boss, Interference 105.531) (Apr. 6, 1983). Minutes from Markman Hearing, Centocor v. Genentech and City of Liu et al., “Cloning and nucleotide sequence of mouse Hope, Case No. CV 08-03573 MRP (CTx) (May 12, 2009). immunoglobuline chain cDNA” Proc. Natl. Acad. Sci. USA 79 Minutes of Order, Centocor v. Genentech and City of Hope, Case No. (24):7852-7856 (Dec. 1982). CV 08-03573 MRP (CTx) (Jun. 9, 2009). Marked-up copy of Mar. 25, 1983 Draft Application (Cabilly Ex. Minutes of Telephonic Status Conference, Centocor v. Genentech 2142, Cabilly v. Boss, Interference 105.531) (Mar. 25, 1983). and City of Hope, Case No. CV 08-03573 MRP (CTx) (Jun. 10, Marked-up copy of proposed Ex. 2122, forwarded to Boss by Cabilly 2009). with Draft Joint Submission Responding to Paper 80, showing com Miozzari and Yanofsky, “Translation of the leader region of parison bet. Cabilly Mar. 25, 1983 draft applin. and Cabilly Mar. 31. Escherichia coli tryptophan operon” Journal of Bacteriology 133 1983 draft applin. (Boss Exhibit 1036, Cabilly v. Boss, Int. 105,531) (3): 1457-1466 (1978). (Mar. 31, 1983). Monoclonal Antibodies, Roger H. Kennett et al., New York: Plenum Marston, “The purification of eukaryotic polypeptides synthesized in Press pp. 8-11, 75-99, 171-182, 275-289 (1980). Escherichia coli" Biochemical Journal 240: 1-12 (1986). Morrison and Schlom, "Recombinant chimeric monoclonal antibod Martens et al., “Heavy chain genes of rabbit IgG: isolation of a cDNA ies' Important Advances in Oncology 1990, Philadelphia: J.B. Lip encodingy heavy chain and identification of two genomic Cygenes” pincott Company pp. 3-18 (1990). Proc. Natl. Acad. Sci. USA 79 (19):60 18-6022 (Oct. 1982). Morrison et al., “Production of novel immunoglobulin molecules by McCarty, M.. “Chemical Nature and Biological Specificity of The gene transfection” Mount Sinai Journal of Medicine 53 (3): 175-180 Substance Inducing Transformation of Pneumococcal Types' Bacte (Mar. 1986). riological Reviews 10 (1-2): 63-71 (Mar. 1946). Mushinski et al., “Mouse immunoglobulin D: construction and char McCormick et al., “Inducible expression of amplified human beta acterization of a cloned 6 chain cDNA” Proc. Natl. Acad. Sci. USA 77 (12):7405-7409 (Dec. 1980). interferon genes in CHO cells' Molecular & Cellular Biology 4 Myers and Spiegelman, "Sodium pyrophosphate inhibition of RNA. (1):166-172 (Jan. 1984). DNA hybrid degradation by reverse transcriptase' Proc. Natl. Acad. MedAdNews (Top 50 pharmaceutical companies charts and lists Sci. USA 75 (11):5329-5333 (Nov. 1978). included) 13 (9): 10, 12, 14 (Sep. 2007). Nakamura, R., “Monoclonal antibodies: methods and clinical labo Medimmune, Inc. v. Genentech, Inc., City of Hope and Celltech, ratory applications' Clinical Physiology & Biochemistry 1 (2-5) : Order remanding to district court (Fed. Cir. Nos. 04-1300,-1364 160-172 (1983). (Mar. 7, 2007). Neumaier et al., “Cloning of the genes for T84.66, an antibody that Medimmune, Inc. v. Genentech, Inc., City of Hope and Celltech, has a high specificity and affinity for carcinoembryonic antigen, and Stipulation and Order Rescheduling Apr. 17, 2007 Status Conference expression of chimeric human/mouse T84.66 genes in myeloma and (C.D. Cal. No. CV 03-2567 MRP (CTx)) (Apr. 12, 2007). Chinese hamster ovary cells' Cancer Research 50 (7) :2128-2134 MedImmune, Inc. v. Genentech, Inc., No. 05-608 (U.S.), slip opinion (Apr. 1, 1990). (Jan. 9, 2007). Newman et al., “Selection and properties of a mouse L-cell MedImmune, Inc.'s Responses and Objections to Genentech, Inc.'s transformant expressing human transferrin receptor' Nature 304 Second Set of Interrogatories (Medimmune v. 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Olsson, L., “Monoclonal antibodies inclinical immunobiology. Deri Pluckthun and Skerra, “Expression of functional antibody Fv and Fab vation, potential, and limitations' Allergy 38 (3) : 145-154 (Apr. fragments in Escherichia coli'Methods in Enzymology 178:497-515 1983). (1989). Order—Motion Times—Bd.R. 104(c) (Cabilly v. Boss, Interference Pluckthun, A., “Escherichia coli Producing Recombinant Antibod 105,531) (Mar. 9, 2007). ies' Recombinant Microbes for Industrial and Agricultural Applica Order—Oral Argument and Other Matters (Cabilly v. Boss, Interfer tions (Part of Bioprocess Technology Series v. 19), Murooka and ence 105,531) (Oct. 3, 2008). Imanaka, New York: Marcel Dekkeer, Inc., Chapter 13, pp. 233-252 Order—Priority Motion Times—Bd.R. 104(c) (Paper No. 90) (1994). (Cabilly v. Boss, Interference 105.531) (Apr. 16, 2008). Polisky, B., "ColE1 replication control circuitry: sense from antisense” Cell 55 (6):929-932 (1988). Order Dismissing Case (GlaxoSmithKline v. Genentech and City of Poljak et al., “Structure of Fab' New at 6A resolution” Nature New Hope, Civil Action 09-61608) (Feb. 17, 2010). Biology 235:137-140 (Feb. 2, 1972). Order Regarding Resolution of Interference, Genentech, Inc. v. Poynton and Reading, "Monoclonal antibodies: the possibilities for Celltech Therapeutics, Ltd., Case No. C98-3926 MMC (Cabilly Ex. cancer therapy” Experimental Biology 43 (1): 13-33 (1984). 2083, Cabilly v. Boss, Interference 105.531) (Mar. 16, 2001). Preliminary Amendment in the Moore U.S. Appl. No. 08/461,071 pp. Overhead slides presented by Dr. Heyneker (Cabilly Ex. 2133, 1-4 (Jun. 5, 1995). Cabilly v. Boss, Interference 105,531) (Feb. 1, 1983). Preliminary Statement of the Party Cabilly et al., Boss et al. v. Cabilly Overhead slides presented by Dr. Heyneker with Kate Murashige's et al., Interference 102,572 (Boss Exhibit 1049, Cabilly v. Boss, notes (Cabilly Exhibit 2183, Cabilly v. Boss, Interference No. Interference 105,531) (Jun. 4, 1991). 105,531) (Feb. 1, 1983). Prentice et al., “Use of anti-T-cell monoclonal antibody OKT3 to Paabo et al., “Association between transplantation antigens and a prevent acute graft-versus-host disease in allogeneic bone-marrow viral membrane protein synthesized from a mammalian expression transplantation for acute leukaemia' Lancet 1 (8274):700-703 (Mar. vector Cell 35 (2):445-453 (Jun. 1983). 27, 1982). Page 57 from Notebook of Dr. Ronald Wetzel, provided to Cabilly Proposed Order Granting Glaxo Group Limited and SmithKline during deposition of Dr. Wetzel on Aug. 5, 2008 (Boss Exhibit 1037. Beecham Corporation d/b/a GlaxoSmithKline's Unopposed Motion Cabilly v. Boss, Interference 105,531) (Feb. 24, 1983). for Enlargement of Time to Respond to Motion to Dismiss or in the Pages from Dr. Ronald Wetzel's Lab Notebook compiled during Alternative Transfer Action (GlaxoSmithKline v. Genentech and City deposition of Dr. Wetzel on Aug. 5, 2008, provided to Cabilly during of Hope, Civil Action 09-61608) (Dec. 23, 2009). deposition of Dr. Wetzel on Aug. 5, 2008 (Boss Exhibit 1038, Cabilly Putnam, F. “Immunoglobulin structure: variability and homology” v. Boss, Interference 105,531) (1983). Science 163 (868):633-643 (Feb. 14, 1969). Pavlakis and Hamer, "Regulation of a metallothionein-growth hor Ratzkin et al., “Expression in Escherichia coli of biologically active mone hybrid gene in bovine papilloma virus' Proc. Natl. Acad. Sci. enzyme by a DNA sequence coding for the human plasminogen USA 80 (2):397-401 (Jan. 1983). activator urokinase” Proc. Natl. Acad. Sci. USA 78 (6):3313-3317 PCT application No. PCT/GB84/00094 (W084/03712 published (Jun. 1981). Sep. 27, 1984), filed by Boss et al. (Cabilly Exhibit 2194, Cabilly v. Record of Oral Hearing (Oral Hearing Held: Jan. 23, 2008) (Paper Boss, Interference No. 105,531) (Mar. 23, 1984). No. 79) (Cabilly v. Boss, Interference 105.531) (Feb. 11, 2008). Pennica et al., “Cloning and Expression of Human Tissue-type Report on the Filing or Determination of an Action Regarding a Plasminogen Activator cDNA in E. coli' Nature 301 :214-221 Patent or Trademark (GlaxoSmithKline v. Genentech and City of (1983). Hope, Case No. 3:10-cv-00675-JSW) (Feb. 18, 2010). Perry and Wetzel, “Disulfide bond engineered into T4 lysozyme: Report to PTO on the Filing of Action Regarding Patent stabilization of the protein toward thermal inactivation” Science 226 (GlaxoSmithKline v. Genentech and City of Hope, Civil Action (4674):555-557 (Nov. 2, 1984). 09-61608) (Oct. 8, 2009). Perry et al., “Purification of monoclonal antibodies using high per Reporter's Transcript of Proceedings, Markman Hearing, CentocOr formance liquid chromatography (HPLC)” Preparative Biochemis v. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx) try 14 (5):431-447 (1984). (May 12, 2009). 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Genentech and City of Hope, Case No. of interchain disulfide bond formation in immunoglobulin G’ Jour CV 08-03573 MRP (CTx)) (Apr. 15, 2009). nal of Biological Chemistry 249 (17):5633-5641 (Sep. 10, 1974). Ringold et al., “Co-expression and Amplification of Dihydrofolate Piggee, C., “Therapeutic antibodies coming through the pipeline' Reductase cDNA and the Escherichia coli XGPRT Gene in Chinese Analytical Chemistry 80 (7):2305-2310 (Apr. 1, 2008). Hamster Ovary Cells' Journal of Molecular & Applied Genetics 1 Plaintiff Centocor Ortho Biotech Inc. and Third Party Defendants (3):165-175 (1981). Global Pharmaceutical Supply Group, LLC, Centocor Biologics, Rituxan?R) Prescribing Information (Provided to Boss during the LLC and Jom Pharmaceutical Services, Inc.'s First Amended Reply Second Deposition of Dennis Burton, Ph.D., Sep. 24, 2008) (Cabilly to Defendants' Second Amended Counterclaims (Centocor v. Exhibit 2211, Cabilly v. Boss, Interference No. 105,531)(Sep. 2008). 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Russell et al., “Monoclonal antibodies for the diagnosis and treat struction with Exhibits, Centocor v. Genentech and City of Hope, ment of transplant rejection' Annual Review of Medicine 35:63-81 Case No. CV 08-03573 MRP (CTx) (Apr. 28, 2009). (1984). Supplemental Report of Dr. Kathryn Calame (MedImmune, Inc. v. Russell, P. “New approaches to the use of antibodies for immunosup Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP (CTx), pression' Transplantation Proceedings 14(3):506-508 (Sep. 1982). C.D.Cal.)) (Apr. 8, 2008). Sann et al., “Rapid fractionation of serum immunoglobulins by high Supplemental Report of Dr. Laurie H. Glimcher (MedImmune, Inc. v. pressure liquid gel permeation chromatography. Application to rou Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP (CTx), tine serologic procedures” Journal of Immunological Methods 59 C.D.Cal.)) (Apr. 8, 2008). (1): 121-127 (Apr. 15, 1983). Supplemental Report of Dr. Michel C. Nussenzweig (MedImmune, Sarma et al., “The Three-Dimensional Structure at 6A Resolution of Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP a Human YG1 Immunoglobulin Molecule” J. Biological Chem. 246: (CTx), C.D.Cal.)) (2008). 3753-3759 (1971). Supplemental Report of James A. Forstner, Ph.D. (MedImmune, Inc. Satz and Singer, "Differential expression of porcine major v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP histocompatibility DNA sequences introduced into mouse L cells' (CTx), C.D.Cal.)) (Apr. 7, 2008). Molecular & Cellular Biology 3 (11):2006-2016 (Nov. 1983). Supplemental Report of Scott A.M. Chambers, Ph.D. (MedImmune, Schlom et al., “Generation of human monoclonal antibodies reactive Inc. v. Genentech, Inc. and City of Hope (Case No. CV03-2567 MRP with human mammary carcinoma cells' Proc. Natl. Acad. Sci. USA (CTx), C.D.Cal.)) (Apr. 7, 2008). 77 (11):6841-6845 (1980). Supplemental Responses to Genentech's Interrogatories Nos. 16 & Schreier et al., “Multiple differences between the nucleic acid 17 (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 sequences of the IgG2a" and IgG2a' alleles of the mouse” Proc. Natl. MRP (CTx)) (Apr. 21, 2010). Acad. Sci. USA 78 (7):4495-4499 (Jul 1981). Supplemental Submission in Response to Paper No. 3 (Cabilly v. Schrohenloher and Hester, "Reassembly of immunoglobulin M Boss, Interference 105,531) (Apr. 25, 2008). heavy and light chains invitro Scandinavian Journal of Immunology Thaler et al. Medical Immunology, Philadelphia:J.B. Lippincott 5 (6-7):637-646 (1976). Company pp. 3-8 (1977). Second Amended Complaint for Declaratory Judgment, Centocor v. Third Declaration of Dennis Burton, Ph.D. (Boss Exhibit 1045. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx) Cabilly v. Boss, Interference 105.531) (Aug. 28, 2008). (Jul. 2, 2009). Second Supplemental Notice of Related Proceedings (Cabilly v. Timeline of Events Preceding Submission of Boss Claim 49 (Cabilly Boss, Interference 105,531) (Jun. 13, 2008). Ex. 2068, Cabilly v. Boss, Interference 105,531). Segal et al., “The three-dimensional structure of a Tissue Culture. Methods and Applications, Kruse, Jr. and Patterson, phosphorylcholine-binding mouse immunoglobulin Fab and the Jr., New York: Academic Press pp. 72-122 (1973). nature of the antigen binding site' Proc. Natl. Acad. Sci. USA 71 Transcript of Deposition of Jeanne Perry (Cabilly Exhibit 2184, (11):4298-4302 (Nov. 1974). Cabilly v. Boss, Interference No. 105.531) (Jan. 18, 2008). Seto et al., “Monoclonal anti-MM46 antibody:ricin A chain conju Transcript of Deposition of Michael Botchan in City of Hope gate: in vitro and in vivo antitumor activity” Cancer Research 42 National Medical Center v. Genentech, Inc., Case No. BC215152 (12):5209-5215 (Dec. 1982). (Los Angeles Co. (Cal.) Super. Ct. (Cabilly Exhibit 2192, Cabilly v. Shulman et al., “A better cell line for making hybridomas secreting Boss, Interference No. 105,531) (May 23, 2001). specific antibodies' Nature 276 (5685):269-270 (Nov. 16, 1978). Transcript of Deposition of Shmuel Cabilly (Cabilly Exhibit 2163, Silverton et al., “Three-dimensional structure of an intact human Cabilly v. Boss, Interference No. 105.531) (Dec. 1, 1999). immunoglobulin’ Proc. Natl. Acad. Sci. USA 74:5140-5144 (1977). Transcript of Oral Hearing on Nov. 13, 2008 (Part 1) (Paper No. 144) Simmons, L. et al., “Expression of full-length immunoglobulins in (Cabilly v. Boss, Interference 105,531) (Jan. 7, 2009). Escherichia coli: Rapid and efficient production of aglycosylated Transcript of Oral Hearing on Nov. 13, 2008 (Part2) (Paper No. 145) antibodies”,Journal of Immunological Methods 263:133-147 (2002). (Cabilly v. Boss, Interference 105,531) (Jan. 7, 2009). Sire et al., “Rat immunoglobulin delta heavy chain gene: nucleotide Transcript of Proceedings before Honorable Mariana Pfaelzer, sequence derived from cloned cDNA” Gene 20 (3):377-386 (Dec. United States District Judge, Markman Hearing, MedImmune, Inc. v. 1982). Genentech, Inc. (C.D. Cal. No. CV 03.2567 MRP (CTx)) (Jul 11, Skerra, Arne, “Bacterial Expression of Immunoglobulin Fragments' 2007). Current Opinion in Immunology 5:256-262 (1993). Transcript of Second Deposition of Dennis Burton, Ph.D. (Cabilly Statement of Material Facts and Responses Thereto (In Motion 4. Exhibit 2212, Cabilly v. Boss, Interference No. 105,531) (Sep. 24, Opposition 4, and Reply 4) (Cabilly v. Boss, Interference 105.531) 2008). (Oct 24, 2008). Travel request (Cabilly Exhibit 2167. Cabilly v. Boss, Interference Statement of Material Facts and Responses Thereto (In Motion 5, No. 105,531) (Mar. 10, 1983). Opposition 5, and Reply 5) (Cabilly v. Boss, Interference 105.531) Tsukada et al., “Effect of a conjugate of daunomycin and antibodies (Oct. 24, 2008). to rat O-fetoprotein on the growth of O-fetoprotein-producing tumor Status Report Regarding Notice of Intent to Issue Ex Parte Reexami cells' Proc. Natl. Acad. Sci. USA 79 (2):621-625 (Jan. 1982). nation Certificate, Centocor v. Genentech and City of Hope, Case No. Tuite et al., “Regulated high efficiency expression of human inter CV 08-03573 MRP (CTx) (Feb. 24, 2009). feron-C. in Saccharomyces cerevisiae 'EMBOJournal 1 (5):603-608 Stepien et al., “Synthesis of a Human Insulin Gene VI. Expression of (1982). the synthetic proinsulin gene in yeast' Gene 24 (2-3):289-297 (Oct. Turner and Rowe, 'Antibodies of IgA and IgG class in normal human 1983). urine” Immunology 12(6):689-699 (Jun. 1967). Stinchcomb et al., “Isolation and characterisation of a yeast chromo Tutorial Declaration of Dennis R. Burton, Ph.D. with Exhibit (Boss somal replicator' Nature 282 (5734):39-43 (Nov. 1, 1979). Exhibit 1016, Cabilly v. Boss, Interference 105,531) (May 25, 2007). Stipulation Re Filing of Amended Complaint to Identify Correct City Tyler et al., “mRNA for Surface immunoglobulin Y chains encodes a of Hope Entity as Defendant, Centocor v. Genentech and City of highly conserved transmembrane sequence and a 28-residue intracel Hope, Case No. CV 08-03573 MRP (CTx) (May 13, 2009). lular domain” Proc. Natl. Acad. Sci. USA 79 (6):2008-2012 (Mar. Sun et al., “Antigen recognition by an antibody light chain' Journal 1982). of Biological Chemistry 269 (1):734–738 (Jan. 7, 1994). U.S. Department of Commerce Patent and Trademark Office, “Chap Supplemental and Amended Responses to Genentech Interrogatories ter 500: Receipt and Handling of Mail and Papers' Manual of Patent (Nos. 13-15 & 18) (Centocor v. Genentech and City of Hope, Case Examining Procedure (Boss Ex. 1044, Cabilly v. Boss, Interference No. CV 08-03573 MRP (CTx)) (Mar. 5, 2010). 105.531), Fourth edition pps.table of contents and 55-70 (Sep. 1982). Supplemental Declaration of Marcus E. Sernel in Support of U.S. Appl. No. 06/358,414 (Moore et al.) (patent application) (Mar. Genentech, Inc.'s and City of Hope's Reply Brief on Claim Con 15, 1982). US 7.923,221 B1 Page 29

U.S. Appl. No. 07/385,102 File History (Plaintiff Exhibit 139, Cheng et al., “Effect of deglycosylation on the binding and Centocor v. Genentech and City of Hope, Case No. CV 08-03573 immunoreactivity of human thyroxine-binding globulin' Journal of MRP (CTx)). Biological Chemistry 254(18):8830-8835 (Sep. 25, 1979). Valenzuela at al., “Synthesis and Assembly of Hepatitis B Virus Civil Minute Order—General (MedImmune, Inc. v. Genentech, Inc., Surface Antigen Particles in Yeast” Nature 298 :347-350 (Jul 22, City of Hope, and Celltech R&D Ltd. pp. 1-4 (Aug. 4, 2003). 1982). Declaration of Dean G. Dunlavey in Support of Defendant Verma et al., “Antibody engineering: comparison of bacterial, yeast, Genentech, Inc.'s Opening Brief Regarding Claim Construction insect and mammalian expression systems' Journal of Immunologi (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Celltech R& D Ltd.) pp. 1-3 with Exhibits A-P (Dec. 19, 2003). cal Methods 216 (1-2):165-181 (Jul. 1, 1998). Declaration of Jeffrey R. Witham in Support of MedImmune, Inc.'s Voller et al., “Enzyme immunoassays with special reference to Opposition Brief in Support of Claim Construction (MedImmune, ELISA techniques' Journal of Clinical Pathology 31 (6):507-520 Inc. v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) pp. 1-3 (Jun. 1978). with Exhibits A-M (Jan. 16, 2004). Ward et al., “Binding Activities of a Repertoire of Single Declaration of Susan L. Friedman in Support of Genentech, Inc.'s Immunoglobulin Variable Domains Secreted from Escherichia coli" Request for Judicial Notice in Support of Reply memorandum for Nature 341:544-546 (Oct. 12, 1989). Motion to Dismiss the Third and Eleventh Causes of Action (MedIm Watson, J. Molecular Biology of the Gene, Menlo Park, CA:W.A. mune, Inc. v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) Benjamin (1976). pp. 1 (Jul. 28, 2003). Weidleet al., “Reconstitution of functionally active antibody directed Defendant City of Hope National Medical Center's Answer (MedIm against creatine kinase from separately expressed heavy and light mune, Inc. v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) chains in non-lymphoid cells' Gene 51 (1):21-29 (1987). pp. 1-37 (Sep. 2, 2003). Weissman et al. Essential Concepts in Immunology, Menlo Park:The Defendant City of Hope's Joinder in Defendant Genentech, Inc.'s Benjamin/Cummings Publishing Company, Inc. pp. 2, 12-19, 23-26, Opening Brief Re Claim Construction (Medlmmune, Inc. v. 49-54 (1978). Genentech, Inc., City of Hope, and Celltech R&D Ltd.) pp. 1-2 (Dec. Weitzman et al., “Mutations in mouse myeloma cells: implications 22, 2003). for human multiple myeloma and the production of Defendant Genentech, Inc.'s Answer and Affirmative Defenses immunoglobulins' Annals of Internal Medicine 85 (1): 110-116 (Jul. (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Celltech 1976). R&D Ltd.) pp. 1-32 (Sep. 2, 2003). Wellborn et al., “Spawning New Forms of Life; Now the Payoff Defendant Genentech, Inc.'s Opening Brief Regarding Claim Con Starts' U.S. News & World Report (Found on p. 48 of Special Report struction (MedImmune, Inc. v. Genentech, Inc., City of Hope, and section.) pp. 1-5 (Mar. 28, 1983). Celltech R&D Ltd.) pp. 1-39 (Dec. 22, 2003). Wetzel et al., “Production of Biologically Active Genentech Inc.'s Notice of Motion and Motion to Dismiss the Third Not-Desacetylthymosino. 1 in Escherichia coli through Expression of and Eleventh Causes of Action; Memorandum of Points and Authori a Chemically Synthesized Gene” Biochemistry 19 :6096-6104 ties in Support (MedImmune, Inc. v. Genentech, Inc., City of Hope, (1980). and Celltech R&D Ltd.) pp. 1-14 (Jun. 4, 2003). Wetzel laboratory notebook 1432 (Cabilly Exhibit 2173, Cabilly v. Genentech, Inc.'s Reply Memorandum of Points and Authorities in Boss, Interference No. 105,531) pp. 26, 59, 70,72, 74, 80 and 81 Support of Motion to Dismiss the Third and Eleventh Causes of (1983). Action (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Wetzel laboratory notebook No. 1432 (Cabilly Ex. 2156, Cabilly v. Celltech R&D Ltd.) pp.-i-v and 1-14 (Jul. 28, 2003). Boss, Interference No. 105,531) pp. 78 (1983). Genentec, Inc.'s Request or Judicial Notice in Support of Reply Wetzel, R., "Applications of Recombinant DNA Technology” Ameri Memorandum for Motion to Dismiss the Third and Eleventh Causes can Scientist 68 (6):664-675 (1980). of Action (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Whitney and Tanford, “Recovery of specific activity after complete Celltech R&D Ltd.) pp. 1-22 (Jul 28, 2003). unfolding and reduction of an antibody fragment” Proc. Natl. Acad. Gillies and Tonegawa, “Expression of cloned immunoglobulin genes Sci. USA 53:524-532 (Mar. 1965). introduced into mouse L cells' Nucleic Acids Research 11 (22) : Wigler et al., "Biochemical transfer of single-copy eucaryotic genes 7981-7997 (Nov. 25, 1983). using total cellular DNA as donor Cell 14:725-731 (1978). Intitial Disclosures of Plaintiff MedImmune, Inc. (MedImmune, Inc. Wigler et al., “Transfer of purified herpes virus thymidine kinase v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) pp. 1-10 gene to cultured mouse cells' Cell 11 (1):223-232 (May 1977). (Aug. 21, 2003). Williams, Jr. et al., “Studies of biologic and serologic activities of Joint Claim Construction Statement (MedImmune, Inc. v. Genentech, rabbit-IgG antibody depleted of carbohydrate residues” Journal of Inc., City of Hope, and Celltech R&D Ltd.) pp. 1-8 (Nov. 7, 2003). Immunology 111 (6):1690-1698 (Dec. 1973). MedImmune, Inc.'s Opposition Brief Regarding Claim Construction Yarmush et al., “Identification and characterization of rabbit-mouse (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Celltech hybridomas secreting rabbit immunoglobulin chains” Proc. Natl. R&D Ltd.) pp. i-ii and 1-38 (Jan. 16, 2004). Acad. Sci. USA 77 (5): 2899-2903 (1980). MedImmune, Inc.'s Responses and Objections to Genentech, Inc.'s Zalcberg and McKenzie, "Hybridomas and monoclonal antibodies: First Set of Interrogatories pp. 1-14 (Sep. 3, 2003). applications in oncology’ Australian & New Zealand Journal of MedImmune, Inc.'s Responses and Objections to Genentech, Inc.'s Surgery 52 (4):431-438 (Aug. 1982). First Set of Requests for Admission (MedImmune, Inc. v. Genentech, Zevalin(R) Prescribing Information (Provided to Boss during the Inc., City of Hope, and Celltech R&D Ltd.) pp. 1-18 (Sep. 3, 2003). Second Deposition of Dennis Burton, Ph.D., Sep. 24, 2008) (Cabilly MedImmune, Inc.'s Responses and Objections to Genentech, Inc.'s Exhibit 2210, Cabilly v. Boss, Interference No. 105,531) (2008). First Set of Requests for the Production of Documents (MedImmune, Amended Memorandum of Decision Re: Defendant Celltech's Inc. v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) pp. Motion for Judgment on the Pleadings and Defendant Genentech's 1-28 (Sep. 3, 2003). Motion for Summary Judgment (MedImmune, Inc. v. Genentech, Memorandum of Decision Re: Defendant Celltech's Motion for Inc., City of Hope, and Celltech R&D Ltd.) pps 1-26 (Jan. 12, 2004). Judgment on the Pleadings and Defendant Genentech's Motion for Celltech R&D Ltd.'s Amended Answer to First Amended Complaint Summary Judgment (MedImmune, Inc. v. Genentech, Inc., City of (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Celltech Hope, and Celltech R&D Ltd.) pp. 1-26 (Dec. 22, 2003). R&D Ltd.) pp. 1-38 (Sep. 22, 2003). Neuberger, M.. “Expression and regulation of immunoglobulin Celltech R&D Ltd.'s Answer (MedImmune, Inc. v Genentech, Inc., heavy chain gene transfected into lymphoid cells' EMBOJournal 2 City of Hope, and Celltech R&D Ltd.) pp. 1-25 (Jun. 4, 2003). (8): 1373-1378 (1983). Celltech R&D Ltd.'s Answer to First Amended Complaint (MedIm Order Granting Genentec Inc.'s Motion to Dismiss the Third and mune, Inc. v. Genentech, Inc., City of Hope, and Celltech R&D Ltd.) Eleventh Causes of Action (MedImmune, Inc. v. Genentech, Inc., City pp. 1-38 (Sep. 2, 2003). of Hope, and Celltech R&D Ltd.) pp. 1-2 (Aug. 11, 2003). US 7.923,221 B1 Page 30

Plaintiff MedImmune, Inc.'s Opposition to Motion by Defendant Order of Dismissal of Entire Action with Prejudice (Centocor v. Genentech, Inc to Dismiss the Third and Eleventh Causes of Action Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) (MedImmune, Inc. v. Genentech, Inc., City of Hope, and Celltech (Sep. 1, 2010). R&D Ltd.) pp. 1-19 (Jul 14, 2003). Reply Memorandum of Points and Authorities in Support of Defen Winkelhake et al., “Effects of pH treatments and deglycosylation of dants Genentech, Inc. and City of Hope's Motion to Preclude or rabbit immunoglobulin G on the binding of Cld'Journal of Biologi Strike Testimony of Dr. Wall (Centocor v. Genentech and City of cal Chemistry 255(7): 2822-2828 (Apr. 10, 1980). Hope, Case No. CV 08-03573 MRP (CTx)) (Aug. 3, 2010). Request for Reexamination under 35 U.S.C. S 302 and 37 C.F.R. Report on the Determination of the Action, Order of Dismissal of 1.510 with Appendices A-D. Entire Action with Prejudice (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Sep. 1, 2010). Centocor Ortho Biotech, Inc.'s and Its Counter-Defendant Affiliates Reporter's Transcript of Proceedings (Status Conference) (Glaxo, et Opposition to Defendants' Motion to Preclude or Strike Testimony of al. v. Genentech, et al., Case No. CV 10-2764-MRP (FMOx)) (Oct Dr. Wall (Centocor v. Genentech and City of Hope, Case No. CV 13, 2010). 08-03573 MRP (CTx)) (Jul 27, 2010). Stipulation of Dismissal of Entire Action with Prejudice (Centocor v. Centocor Ortho Biotech, Inc.'s and Its Counter-Defendant Affiliates Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Reply in Support of Their Motion for Construction of Claim Term (Aug. 30, 2010). “Immunoglobulin' (Motion No. 2) (Centocor v. Genentech and City Transcript of Proceedings, Motions Hearing (Centocor v. Genentech of Hope, Case No. CV 08-03573 MRP (CTx)) (Aug. 3, 2010). and City of Hope, Case No. CV 08-03573 MRP (CTx) (Aug. 17, Centocor Ortho Biotech, Inc.'s and Its Counter-Defendant Affiliates 2010). Reply in Support of Their Motion for Summary Judgment of Antici MedImmune, Inc.'s Responses and Objections to Genentech, Inc.'s pation (Motion No. 5) (Centocor v. Genentech and City of Hope, Second Set of Interrogatories (Feb. 24, 2004). Case No. CV 08-03573 MRP (CTx)) (Aug. 3, 2010). Joint Claim Construction Statement (Nov. 7, 2003). Centocor Ortho Biotech, Inc.'s and Its Counter-Defendant Affiliates Declaration of Dean G. Dunlavey in Support of Defendant Reply in Support of Their Motion for Summary Judgment of Inval Genentech, Inc.'s Opening Brief Regarding Claim Construction with idity of Claim 33 for Failure to Comply with 35 USC 112 (Motion Exhibits A-P (Dec. 22, 2003). No. 4) (Centocor v. Genentech and City of Hope, Case No. CV Supplemental Declaration of Dean G. Dunlavey in Support of Defen 08-03573 MRP (CTx)) (Aug. 3, 2010). dant Genentech, Inc.'s Reply Brief Regarding Claim Construction Centocor Ortho Biotech, Inc.'s and Its Counter-Defendant Affiliates with Exhibit Q (Feb. 13, 2004). Reply in Support of Their Motion for Summary Judgment That Claim Defendant Genentech, Inc.'s Reply Brief Regarding Claim Construc 33 is Invalid for Failure to Disclose the Best Mode (Motion No. 6) tion (Feb. 13, 2004). (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 Deposition Transcript of Genentech through witness, Janet Hasak MRP (CTx)) (Aug. 3, 2010). (Feb. 25, 2004). Genentech, Inc. and City of Hope's Opposition to Centocor Inc.'s Deposition Transcript Exhibits 1-29 of Janet Hasak (Feb. 25, 2004). Motion for Summary Judgment That Claim 33 is Invalid for Failure Deposition Transcript and Exhibits 30-33 of Genentech through wit to Disclose the Best Mode (Motion No. 6) (Centocor v. Genentech ness, Janet Hasak (Feb. 25, 2004). and City of Hope, Case No. CV 08-03573 MRP (CTX)) (Jul. 27. Deposition Transcript of Wendy M. Lee and Exhibits 34-47 (Mar, 4. 2010). 2004). Genentech, Inc. and City of Hope's Opposition to Centocor's Motion Deposition Transcript of Wendy M. Lee and Exhibits 48-60 (Mar. 5, for Summary Judgment of Invalidity of Claim 33 for Failure to 2004). Comply with 35 USC 112 (Motion No. 4) (Centocor v. Genentech Joint Statement Responsive to Court's Jan. 28, 2004 Order re: Terms and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Jul 27, to be Construed at Markman Hearing (Feb. 9, 2004). 2010). Expert Report of Carlo M. Croce (Centocor v. Genentech and City of Memorandum in Support of Centocor Ortho Biotech, Inc.'s and Its Hope, Case No, CV 08-03573 MRP (CTx)) (Jun. 4, 2010). Counter-Defendant Affiliates Motion for Construction of Claim Expert Report of Matthew P. Scott (Centocor v. Genentech and City Term “Immunoglobulin' (Motion No. 2) (Centocor v. Genentech and of Hope, Case No. CV 08-03573 MRP (CTx)) (Jun. 4, 2010). City of Hope, Case No. CV 08-03573 MRP (CTx) (Jul 12, 2010). Expert Report of Robert B. Freedman (Centocor v. Genentech and Memorandum in Support of Centocor Ortho Biotech, Inc.'s and Its City of Hope, Case No. CV 08-03573 MRP (CTx)) (Jun. 4, 2010). Counter-Defendant Affiliates' Motion for Summary Judgment of Oral deposition of Eugene C. RZuoidlo (Centocor v. Genentech and Anticipation (Motion No. 5) (Centocor v. Genentech and City of City of Hope, Case No. CV 08-03573 MRP (CTX)) (Jun. 25, 2010). Hope, Case No. CV 08-03573 MRP (CTx)) (Jul 12, 2010). Response Expert Report of Mark E. Nusbaum (Centocor v. Memorandum in Support of Centocor Ortho Biotech, Inc.'s and Its Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Counter-Defendant Affiliates' Motion for Summary Judgment of (Jun. 4, 2010). Invalidity of Claim 33 for Failure to Comply with 35 USC 112 Videotaped Deposition of Arne Skerra (Centocor v. Genentech and (Motion No. 4) (Centocor v. Genentech and City of Hope, Case No. City of Hope, Case No. CV 08-03573 MRP (CTX)) (Jun. 24, 2010). CV 08-03573 MRP (CTx) (Jul 12, 2010). Videotaped Deposition of Carlo M. Croce (Centocor v. Genentech Memorandum in Support of Centocor Ortho Biotech, Inc.'s and Its and City of Hope, Case No. CV 08-03573 MRP (CTX)) (Jul. 8, 2010). Counter-Defendant Affiliates' Motion for Summary Judgment That Videotaped Deposition of Daniel G.Yansura (Centocor v. Genentech Claim33 is Invalid for Failure to Disclose the Best Mode (MotionNo. and City of Hope, Case No. CV 08-03573 MRP (CTx)) (Apr. 20, 6) (Centocor v. Genentech and City of Hope, Case No. CV 08-03573 2010). MRP (CTx) (Jul 12, 2010). Videotaped Deposition of Jeffrey Kushan (Centocor v. Genentech Memorandum of Points and Authorities in Support of Defendants and City of Hope, Case No. CV 08-03573 MRP (Ctx)) (Jun. 1, 2010). Genentech, Inc. and City of Hope's Motion to Preclude or Strike Videotaped Deposition of Mark E. Nusbaum (Centocor v. Genentech Testimony of Dr. Wall (Centocor v. Genentech and City of Hope, and City of Hope, Case. No. CV 08-03573 MRP (CTx)) (Jun. 22, Case No. CV 08-03573 MRP (CTx)) (Jul 12, 2010). 2010). Opposition by Genentech, Inc. and City of Hope to Centocor Ortho Videotaped Deposition of Mark X. Sliwkowski (Centocor v. Biotech, Inc.'s and Its Counter-Defendant Affiliates' Motion for Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Summary Judgment of Anticipation (Motion No. 5) (Centocor v. (Apr. 30, 2010). Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Videotaped Deposition of Matthew Peter Scott (Centocor v. (Jul 27, 2010). Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) Opposition of Genentech, Inc. & City of Hope to Centocor Ortho (Jun. 15, 2010). Biotech, Inc.'s Motion for Construction of Claim Term Videotaped Deposition of Robert B. Freedman (Centocorv. “Immunoglobulin' (Motion No. 2) (Centocor v. Genentech and City Genentech and City of Hope, Case No. CV 08-03573 MRP (CTx)) of Hope, Case No. CV 08-03573 MRP (CTx)) (Jul. 27, 2010). (Jun. 22, 2010). US 7.923,221 B1 Page 31

Videotaped Deposition of Sharon Elizabeth Crane (Centocor v. Maniatis et al. Molecular Cloning A Laboratory Manual, New Genentech and City of Hope, Case No. CV 08-03573 MRP (CTX)) York: Cold Spring Harbor Laboratory pp. 1-545 (1982). (Jun. 10, 2010). Nagahariat al., “Control of tryptophan synthetase amplified by vary Request for Reexamination filed on May 13, 2005 for U.S. PatentNo. ing the numbers of composite plasmids in Escherichia coli cells' 6,331.415 with Appendices 1-21. Gene 1 (2): 141-152 (Mar. 1977). Restriction Requirement (Paper No. 4) dated Mar. 8, 1990 in U.S. Nagahari et al., “Derepression of E. coli trp operon on interfamilial Appl. No. 07/205,419. transfer” Nature 266 (5604):745-746 (Apr. 21, 1977). Restriction Requirement (Paper No. 11) dated Sep. 7, 1990 in U.S Nagahari et al., “Expression of Escherichia coli tryptophan operon in Appl. No. 07/205,419. Rhizobium leguminosarum" Mol. Gen. Genet. 171 (2) : 115-119 Interview Summary (Paper No. 22) in U.S. Appl. No. 07/205,419. (Mar. 20, 1979). Amendment After Interference (Paper No. 24) filed Oct. 4, 2001 in Pauza et al., “Genes encoding Escherichia coli aspartate U.S. Appl. No. 07/205,419. transcarbamoylase: the pyrB-pyrI operon” Proc. Natl. Acad. Sci. File History of U.S. Patent No. 4,816,397 (Boss et al.). USA 79 (13):4020-4024 (Jul 1982). Aiba et al., “New approach to tryptophan production by Escherichia Plaintiffs and counterclaim defendants' preliminary contentions coli: genetic manipulation of composite plasmids in vitro' Appl. regarding the invalidity of U.S. Patent No. 6,331.415 (Glaxo et al. v. Environ. Microbiol. 43 (2):289-297 (Feb. 1982). Genentech and City of Hope, Case No. CV-10-02764 MRP (FMOx), Brandsma et al., “Effect of lexA and Ssb genes, present on a uvrA Exhibits A & Battached.) (Dec 13, 2010). recombinant plasmid, on the UV survival of Escherichia coli K-12” Rapoport et al., “Construction of a colony bank of E. coli containing Gene 18(1):77-85 (Apr. 1982). hybrid plasmids representative of the Bacillus subtilis 168 genome. Enger-Valk et al., "Construction of new cloning vehicles with genes Expression of functions harbored by the recombinant plasmids in B. of the tryptophan operon of Escherichia coli as genetic markers' subtilis” Mol. Gen. Genet. 176 (2): 239-245 (Oct. 3, 1979). Gene 9(1-2): 69-85 (Apr. 1980). Roof et al., “The organization and regulation of the pyrBI operon in Enger-Valk et al., “The construction of new vehicles for the cloning E. coli includes a rho-independent attenuator sequence' Mol. Gen. of transcription termination signals' Nucleic Acids Research 9 (8): Genet. 187 (3):391-400 (1982). 1973-1989 (Apr. 1981). Turnbough Jr., C., “Regulation of Escherichia coli aspartate Fiandt et al., “Physical mapping of the trp endpoint in the n-tL transcarbamylase synthesis by guanosine tetraphosphate and segment of phage lambda trpE-A' Virology 61 (1) :312-314 (Sep. pyrimidine ribonucleoside triphosphates' J. Bacteriol. 153 (2):998 1974). 1007 (Feb. 1983). Friesen and An, "Expression vehicles used in recombinant DNA Turnbough, Jr. et al., “Attenuation control of pyrBI operon expression technology” Biotechnol. Adv. 1 (2):205-227 (1983). in Escherichia coli K-12” Proc. Natl. Acad. Sci. USA 80 (2):368-372 Gough and Murray, "Expression of the hepatitis B virus Surface, core (Jan. 1983). and E antigen genes by stable rat and mouse cell lines' J. Mol. Biol. van Leerdam et al., “Cloning of both ends and the thermo-inducible 162(1):43-67 (Nov. 25, 1982). genes A and B ofbacteriophage Mu on a multicopy plasmid' Gene 13 Hallewell and Emtage, "Plasmid vectors containing the tryptophan (1):111-114 (Jan.-Feb. 1981). operon promoter Suitable for efficient regulated expression of foreign Wagner et al., “Transport of hemolysin across the outer membrane of genes' Gene 9 (1-2):27-47 (Apr. 1980). Escherichia coli requires two functions' J. Bacteriol. 154 (1):200 Hershfield et al., “Plasmid ColEl as a molecular vehicle for cloning 210 (Apr. 1983). and amplification of DNA” Proc. Natl. Acad. Sci. USA 71 (9):3455 Watson et al. Recombinant DNA-A Short Course, New 3459 (Sep. 1974). York:Scientific American Books (W.H. Freeman & Company) pp. Horowitz and Platt, “Identification of trp-p2, and internal promoter in 1-260 (1983). the tryptophan operon of Escherichia coli'J. Mol. Biol. 156 (2): Wild at al., “A mutation in the catalytic cistron of aspartate 257-267 (Apr. 5, 1982). carbamoyltransferase affecting catalysis, regulatory response and Imamoto and Tani, “Diversity of regulation of genetic transcription' holoenzyme assembly' Nature 292 (5821):373-375 (Jul 23, 1981). Nat. New Biol. 240 (101): 172-175 (Dec. 6, 1972). Williams et al., “Expression of Escherichia coli trp genes and the Kaufman and Sharp, Amplification and Expression of Sequences mouse dihydrofolate reductase gene cloned in Bacillus subtilis' Cotransfected with a Modular Dihydrofolate Reductase Comple Gene 16 (1-3): 199-206 (Dec. 1981). mentary DNA Gene” J. Mol. Biol. 159 (4):601-621 (Aug. 25, 1982). Yamamoto and Imamoto, “Differential stability of trp messenger Legrain et al., “Structural and regulatory mutations allowing utiliza RNA synthesized originating at the trp promoter and pL promoter of tion of citrulline or carbamoylaspartate as a source of lambda trp phage” J. Mol. Biol.92 (2):289-304 (Feb. 25, 1975). carbamoylphosphate in Escherichia coli K-12” J. Bacteriol. 128 (1):39-48 (Oct. 1976). * cited by examiner U.S. Patent Apr. 12, 2011 Sheet 1 of 19 US 7.923,221 B1

Fab' fragment

SSSS SSSS Fc fragment

U.S. Patent Apr. 12, 2011 Sheet 10 of 19 US 7.923,221 B1

xial EcoR

Psi s Bornhi Sc TR Avo

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EcoR Kenow Pli Klenow4 NTPs Pl + 4 NPs Pst Pst solo e ACEA isolate torge vector frogment frogment T4 ONA (lgase

parCEA trip2O7 U.S. Patent Apr. 12, 2011 Sheet 11 of 19 US 7.923,221 B1

Xbo EcoR EcoR geed So Ps TR Born Ap So Ter Avo

Pst

isolo te v 850 bp fragment Avo Taq 1, Barnh! Avo Kenow Pot solo te 500 bp frogment Klenow Pol 1 + 4 d NTP's + 4 d NTP's Toq I Born H solote 375bp Avoi, solote lorge Taq fragment vector fragment T4 DNA ligose

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U.S. Patent Apr. 12, 2011 Sheet 12 of 19 US 7.923,221 B1

Fig.8A. U.S. Patent Apr. 12, 2011 Sheet 13 of 19 US 7.923,221 B1

U.S. Patent Apr. 12, 2011 Sheet 14 of 19 US 7.923,221 B1

Fig.8C.

U.S. Patent Apr. 12, 2011 Sheet 16 of 19 US 7.923,221 B1

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O IOO OOO OOOO Concentration (ng/ml) Fig. 70 U.S. Patent Apr. 12, 2011 Sheet 17 of 19 US 7.923,221 B1

containing humon 2 heavy chain n EcoRI cDNA insert Y Ban Hl v Sali

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Born H TcR Sol R Sall o Pyu • Wool • Any restriction enzyme which • Klenow Poll dNTP's cuts in variable region v Py • Kenow Poll + d NTP's isolate vector fragment solate antibody fragment T4 DNA ligase

DELETE AS FOLLOWS: Clone Xbal to Pvull fragment in M3 pintermediote o/n vitro site-directed humon/mouse deletion mutagenesis hybrid (XAP) Avoll (see Adelman et al., DNA 2, 183 (1983)) • Clone modified Xbo to Pvul Bonn H. fragment back in Sal p-Intermediate human/mouse hybrid (XAP)

pCEAtrip2O7 Fig 12. Ap humon/mouse Avoli hybrid (XAP) (pChimi) Born Tc" sai U.S. Patent Apr. 12, 2011 Sheet 19 Of 19 US 7.923,221 B1

US 7,923,221 B1 1. 2 METHODS OF MAKING ANTIBODY HEAVY Immunol. 6: 511 (1976)). In this process, splenocytes or AND LIGHT CHANS HAVING SPECIFICITY lymphocytes from a mammal which has been injected with FORADESIRED ANTIGEN antigenarefused with a tumor cell line, thus producing hybrid cells or “hybridomas’ which are both immortal and capable This is a continuation of application(s) Ser. No. 07/205,419 of producing the genetically coded antibody of the B cell. The filed on 10 Jun. 1988, now U.S. Pat. No. 6,331415, issued on hybrids thus formed are segregated into single genetic strains 18 Dec. 2001, which is a continuation of Ser. No. 06/483,457 by selection, dilution, and regrowth, and each strain thus filed on 8 Apr. 1983, now U.S. Pat. No. 4,816,567, issued on represents a single genetic line. They therefore produce 28 Mar. 1989, which applications are incorporated herein by immunoreactive antibodies against a desired antigen which reference and to which application(s) priority is claimed 10 are assured to be homogenous, and which antibodies, refer under 35 USC S120. encing their pure genetic parentage, are called "monoclonal'. BACKGROUND OF THE INVENTION Hybridoma technology has to this time been focused largely on the fusion of murine lines, but human-human hybridomas This invention relates to the field of immunoglobulin pro 15 (Olsson, L. et al., Proc. Natl. Acad. Sci. (USA), 77: 5429 duction and to modification of naturally occurring immuno (1980)); human-murine hybridomas (Schlom, J., et al. (ibid) globulin amino acid sequences. Specifically, the invention 77: 6841 (1980)) and several other xenogenic hybrid combi relates to using recombinant techniques to produce both nations have been prepared as well. Alternatively, primary, immunoglobulins which are analogous to those normally antibody producing, B cells have been immortalized in vitro found in Vertebrate systems and to take advantage of these by transformation with viral DNA. gene modification techniques to construct chimeric or other Polyclonal, or, much more preferably, monoclonal, anti modified forms. bodies have a variety of useful properties similar to those of A. Immunoglobulins and Antibodies the present invention. For example, they can be used as spe Antibodies are specific immunoglobulin polypeptides pro cific immunoprecipitating reagents to detect the presence of duced by the vertebrate immune system in response to chal 25 the antigen which elicited the initial processing of the B cell lenge by foreign proteins, glycoproteins, cells, or other anti genome by coupling this antigen-antibody reaction with Suit genic foreign Substances. The sequence of events which able detection techniques such as labeling with radioisotopes permits the organism to overcome invasion by foreign cells or or with enzymes capable of assay (RIA, EMIT, and ELISA). to rid the system of foreign Substances is at least partially Antibodies are thus the foundation of immuno diagnostic understood. An important part of this process is the manufac 30 tests for many antigenic Substances. In another important use, ture of antibodies which bind specifically to a particular for antibodies can be directly injected into Subjects Suffering eign substance. The binding specificity of such polypeptides from an attack by a substance or organism containing the to a particular antigen is highly refined, and the multitude of antigen in question to combat this attack. This process is specificities capable of being generated by the individual currently in its experimental stages, but its potential is clearly vertebrate is remarkable in its complexity and variability. 35 seen. Third, whole body diagnosis and treatment is made Thousands of antigens are capable of eliciting responses, possible because injected antibodies are directed to specific each almost exclusively directed to the particular antigen target disease tissues, and thus can be used either to determine which elicited it. the presence of the disease by carrying with them a suitable Immunoglobulins include both antibodies, as above label, or to attack the diseased tissue by carrying a suitable described, and analogous protein Substances which lack anti 40 drug. gen specificity. The latter are produced at low levels by the Monoclonal antibodies produced by hybridomas, while lymph system and in increased levels by myelomas. theoretically effective as suggested above and clearly prefer A.1 Source and Utility able to polyclonal antibodies because of their specificity, Two major sources of vertebrate antibodies are presently suffer from certain disadvantages. First, they tend to be con utilized generation in situ by the mammalian B lympho 45 taminated with other proteins and cellular materials of hybri cytes and in cell culture by B-cell hybrids. Antibodies are doma, (and, therefore, mammalian) origin. These cells con made in situ as a result of the differentiation of immature B tain additional materials, notably nucleic acid fragments, but lymphocytes into plasma cells, which occurs in response to protein fragments as well, which are capable of enhancing, stimulation by specific antigens. In the undifferentiated B causing, or mediating carcinogic responses. Second, hybri cell, the portions of DNA coding for the various regions on the 50 doma lines producing monoclonal antibodies tend to be immunoglobulin chains are separated in the genomic DNA. unstable and may alter the structure of antibody produced or The sequences are reassembled sequentially prior to tran stop producing antibody altogether (Kohler, G., et al., Proc. Scription. A review of this process has been given by Gough, Natl. Acad. Sci (USA) 77: 2197 (1980); Morrison, S. L., J. Trends in Biochem Sci, 6: 203 (1981). The resulting rear Immunol. 123: 793 (1979)). The cell line genome appears to ranged genome is capable of expression in the mature B 55 alter itself in response to stimuli whose nature is not currently lymphocyte to produce the desired antibody. Even when only known, and this alteration may result in production of incor a single antigen is introduced into the sphere of the immune rect sequences. Third, both hybridoma and B cells inevitably system for a particular mammal, however, a uniform popula produce certain antibodies in glycosylated form (Melchers, tion of antibodies does not result. The in situ immune F., Biochemistry, 10: 653 (1971)) which, under some circum response to any particular antigen is defined by the mosaic of 60 stances, may be undesirable. Fourth, production of both responses to the various determinants which are present on monoclonal and polyclonal antibodies is relatively expensive. the antigen. Each Subset of homologous antibody is contrib Fifth, and perhaps most important, production by current uted by a single population of B cells—hence in situ genera techniques (either by hybridoma or by B cell response) does tion of antibodies is “polyclonal'. not permit manipulation of the genome so as to produce This limited but inherent heterogeneity has been overcome 65 antibodies with more effective design components than those in numerous particular cases by use of hybridoma technology normally elicited in response to antigens from the mature B to create “monoclonal antibodies (Kohler, et al., Eur: J. cell in situ. The antibodies of the present invention do not US 7,923,221 B1 3 4 suffer from the foregoing drawbacks, and, furthermore, offer Clinical Immunobiology pp 1-18, W. B. Sanders (1980); the opportunity to provide molecules of Superior design. Kohl, S., et al., Immunology, 48: 187 (1983)); while the vari Even those immunoglobulins which lack the specificity of able region determines the antigen with which it will react. antibodies are useful, although over a smaller spectrum of potential uses than the antibodies themselves. In presently 5 B. Recombinant DNA Technology understood applications, such immunoglobulins are helpful in protein replacement therapy for globulin related anemia. In Recombinant DNA technology has reached sufficient this context, an inability to bind to antigen is in fact helpful, as Sophistication that it includes a repertoire of techniques for the therapeutic value of these proteins would be impaired by cloning and expression of gene sequences. Various DNA Such functionality. At present, such non-specific antibodies 10 sequences can be recombined with some facility, creating are derivable in quantity only from myeloma cell cultures new DNA entities capable of producing heterologous protein suitably induced. The present invention offers an alternative, product in transformed microbes and cell cultures. The gen more economical Source. It also offers the opportunity of eral means and methods for the in vitro ligation of various cancelling out specificity by manipulating the four chains of bluntended or “sticky' ended fragments of DNA, for produc the tetramer separately. 15 ing expression vectors, and for transforming organisms are A.2 General Structure Characteristics now in hand. The basic immunoglobin structural unit in vertebrate sys DNA recombination of the essential elements (i.e., an ori tems is now well understood (Edelman, G. M., Ann. N.Y. gin of replication, one or more phenotypic selection charac Acad. Sci., 190: 5 (1971)). The units are composed of two teristics, expression control sequence, heterologous gene identical light polypeptide chains of molecular weight insert and remainder vector) generally is performed outside approximately 23,000 daltons, and two identical heavy the host cell. The resulting recombinant replicable expression chains of molecular weight 53,000-70,000. The four chains vector, or plasmid, is introduced into cells by transformation are joined by disulfide bonds in a “Y” configuration wherein and large quantities of the recombinant vehicle is obtained by the light chains bracket the heavy chains starting at the mouth growing the transformant. Where the gene is properly of the Y and continuing through the divergent region as shown 25 inserted with reference to portions which govern the tran in FIG.1. The “branch” portion, as there indicated, is desig Scription and translation of the encoded DNA message, the nated the Fab region. Heavy chains are classified as gamma, resulting expression vector is useful to produce the polypep mu, alpha, delta, or epsilon, with some Subclasses among tide sequence for which the inserted gene codes, a process them, and the nature of this chain, as it has a long constant referred to as “expression.” The resulting product may be region, determines the “class of the antibody as IgG, IgM, 30 obtained by lysis, if necessary, of the host cell and recovery of IgA, Ig|D, or IgE. Light chains are classified as eitherkappa or the product by appropriate purifications from other proteins. lambda. Each heavy chain class can be prepared with either In practice, the use of recombinant DNA technology can kappa or lambda light chain. The light and heavy chains are express entirely heterologous polypeptides—so-called direct covalently bonded to each other, and the “tail portions of the expression—or alternatively may express a heterologous two heavy chains are bonded to each other by covalent disul 35 polypeptide fused to a portion of the amino acid sequence of fide linkages when the immunoglobulins are generated either a homologous polypeptide. In the latter cases, the intended by hybridomas or by B cells. However, if non-covalent asso bioactive product is sometimes rendered bioinactive within ciation of the chains can be effected in the correct geometry, the fused, homologous/heterologous polypeptide until it is the aggregate will still be capable of reaction with antigen, or cleaved in an extracellular environment. of utility as a protein Supplement as a non-specific immuno 40 The art of maintaining cell or tissue cultures as well as globulin. microbial systems for studying genetics and cell physiology The amino acid sequence runs from the N-terminal end at is well established. Means and methods are available for the top of the Y to the C-terminal end at the bottom of each maintaining permanent cell lines, prepared by Successive chain. At the N-terminal end is a variable region which is serial transfers from isolated cells. For use in research, such specific for the antigen which elicited it, and is approximately 45 cell lines are maintained on a solid Support in liquid medium, 100 amino acids in length, there being slight variations or by growth in Suspension containing Support nutriments. between light and heavy chain and from antibody to antibody. Scale-up for large preparations seems to pose only mechani The variable region is linked in each chain to a constant region cal problems. which extends the remaining length of the chain. Linkage is seen, at the genomic level, as occurring through a linking 50 SUMMARY OF THE INVENTION sequence known currently as the “J” region in the light chain gene, which encodes about 12 amino acids, and as a combi The invention relates to antibodies and to non-specific nation of "D' region and “J” region in the heavy chain gene, immunoglobulins (NSIs) formed by recombinant techniques which together encode approximately 25 amino acids. using suitable host cell cultures. These antibodies and NSIs The remaining portions of the chain are referred to as 55 can be readily prepared in pure"monoclonal form. They can constant regions and within a particular class do not to vary be manipulated at the genomic level to produce chimeras of with the specificity of the antibody (i.e., the antigen eliciting variants which draw their homology from species which dif it). fer from each other. They can also be manipulated at the As stated above, there are five known Major classes of protein level, since all four chains do not need to be produced constant regions which determine the class of the immuno 60 by the same cell. Thus, there are a number of “types of globulin molecule (IgG, IgM, IgA, Ig), and IgE correspond immunoglobulins encompassed by the invention. ing to Y, L, C, 6, and e heavy chain constant regions). The First, immunoglobulins, particularly antibodies, are pro constant region or class determines Subsequent effector func duced using recombinant techniques which mimic the amino tion of the antibody, including activation of complement (Ka acid sequence of naturally occurring antibodies produced by bat, E. A., Structural Concepts in Immunology and Immu 65 either mammalian B cells in situ, or by B cells fused with nochemistry, 2nd Ed., p. 413–436, Holt, Rinehart, Winston Suitable immortalizing tumor lines, i.e., hybridomas. Second, (1976)), and other cellular responses (Andrews, D. W., et al., the methods of this invention produce, and the invention is US 7,923,221 B1 5 6 directed to, immunoglobulins which comprise polypeptides comprising light and heavy chains usually aggregated in the not hitherto found associated with each other in nature. Such “Y” configuration of FIG. 1, with or without covalent linkage reassembly is particularly useful in producing “hybrid anti between them: “immunoglobulins' refers to such assemblies bodies capable of binding more than one antigen; and in whether or not specific immunoreactive activity is a property. producing "composite' immunoglobulins wherein heavy and “Non-specific immunoglobulin” (“NSI) means those immu light chains of different origins essentially damp out speci noglobulins which do not possess specificity—i.e., those ficity. Third, by genetic manipulation, "chimeric' antibodies which are not antibodies. can be formed wherein, for example, the variable regions "Mammalian antibodies' refers to antibodies wherein the correspond to the amino acid sequence from one mammalian model system, whereas the constant region mimics the amino amino acid sequences of the chains are homologous with acid sequence of another. Again, the derivation of these two 10 those sequences found in antibodies produced by mammalian mimicked sequences may be from different species. Fourth, systems, either in situ, or in hybridomas. These antibodies also by genetic manipulation, “altered antibodies with mimic antibodies which are otherwise capable of being gen improved specificity and other characteristics can be formed. erated, although in impure form, in these traditional systems. Two other types of immunoglobulin-like moieties may be “Hybrid antibodies' refers to antibodies wherein chains produced: “univalent antibodies, which are useful as homing 15 are separately homologous with referenced mammalian anti carriers to target tissues, and “Fab proteins” which include body chains and represent novel assemblies of them, so that only the “Fab' region of an immunoglobulin molecule i.e., the two different antigens are precipitable by the tetramer. In branches of the “Y”. These univalent antibodies and Fab hybrid antibodies, one pair of heavy and light chain is fragments may also be “mammalian’ i.e., mimic mammalian homologous to antibodies raised against one antigen, while amino acid sequences; novel assemblies of mammalian the other pair of heavy and light chain is homologous to those chains, or chimeric, where for example, the constant and raised against another antigen. This results in the property of variable sequence patterns may be of different origin. Finally, "divalence' i.e., ability to bind two antigens simultaneously. either the light chain or heavy chain alone, orportions thereof, Such hybrids may, of course, also be formed using chimeric produced by recombinant techniques are included in the chains, as set forth below. invention and may be mammalian or chimeric. 25 “Composite' immunoglobulins means those wherein the In other aspects, the invention is directed to DNA which heavy and light chains mimic those of different species ori encodes the aforementioned NSIs, antibodies, and portions thereof, as well as expression vectors or plasmids capable of gins or specificities, and the resultant is thus likely to be a effecting the production of such immunoglobulins in Suitable non-specific immunoglobulin (NSI), i.e. —lacking in anti host cells. It includes the host cells and cell cultures which body character. result from transformation with these vectors. Finally, the 30 “Chimeric antibodies' refers to those antibodies wherein invention is directed to methods of producing these NSIs and one portion of each of the amino acid sequences of heavy and antibodies, and the DNA sequences, plasmids, and trans light chains is homologous to corresponding sequences in formed cells intermediate to them. antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chains is BRIEF DESCRIPTION OF THE DRAWINGS 35 homologous to corresponding sequences in another. Typi cally, in these chimericantibodies, the variable region of both FIG. 1 is a representation of the general structure of immu light and heavy chains mimics the variable regions of anti noglobulins. bodies derived from one species of mammals, while the con FIGS. 2A-B show the detailed sequence of the cDNA insert stant portions are homologous to the sequences in antibodies of pK17G4 which encodes kappa anti CEA chain. 40 derived from another. One clear advantage to Such chimeric FIG.3 shows the coding sequence of the fragment shown in forms is that, for example, the variable regions can conve FIG. 2, along with the corresponding amino acid sequence. niently be derived from presently known sources using FIGS. 4A-C show the combined detailed sequence of the readily available hybridomas or B cells from nonhuman host cDNA inserts of py298 and py11 which encode gamma anti organisms in combination with constant regions derived CEA chain. 45 from, for example, human cell preparations. While the vari FIGS.5A-B show the corresponding amino acid sequence able region has the advantage of ease of preparation, and the encoded by the fragment in FIG. 4. specificity is not affected by its source, the constant region FIGS. 6 and 7 outline the construction of expression vec being human, is less likely to elicit an immune response from tors for kappa and gamma anti-CEA chains respectively. a human Subject when the antibodies are injected than would FIGS. 8A, 8B, and 8C show the results of sizing gels run on 50 the constant region from a non-human source. extracts of E. coli expressing the genes for gamma chain, However, the definition is not limited to this particular kappa chain, and both kappa and gamma chains respectively. example. It includes any antibody in which either or both of FIG.9 shows the results of western blots of extracts of cells the heavy or light chains are composed of combinations of transformed as those in FIG. 8. sequences mimicking the sequences in antibodies of different FIG. 10 shows a standard curve for ELISA assay of anti 55 Sources, whether these sources be differing classes, differing CEA activity. antigen responses, or differing species of origin and whether FIGS. 11 and 12 show the construction of a plasmid for or not the fusion point is at the variable/constant boundary. expression of the gene encoding a chimeric heavy chain. Thus, it is possible to produce antibodies in which neither the FIG. 13 shows the construction of a plasmid for expression constant nor the variable region mimic known antibody of the gene encoding the Fab region of heavy chain. 60 sequences. It then becomes possible, for example, to con struct antibodies whose variable region has a higher specific DETAILED DESCRIPTION affinity for a particular antigen, or whose constant region can elicit enhanced complement fixation or to make other A. Definitions improvements in properties possessed by a particular con 65 stant region. As used herein, “antibodies' refers to tetramers or aggre “Altered antibodies' means antibodies wherein the amino gates thereof which have specific immunoreactive activity, acid sequence has been varied from that of a mammalian or US 7,923,221 B1 7 8 other vertebrate antibody. Because of the relevance of recom cable in the host organisms eitheras episomes or as an integral binant DNA techniques to this invention, one need not be part of the chromosomal DNA. Clearly a lack of replicability confined to the sequences of amino acids found in natural would render them effectively inoperable. A useful, but not a antibodies; antibodies can be redesigned to obtain desired necessary, element of an effective expression vector is a characteristics. The possible variations are many and range 5 marker encoding sequence—i.e. a sequence encoding a pro from the changing of just one or a few amino acids to the tein which results in a phenotypic property (e.g. tetracycline complete redesign of for example, the constant region. resistance) of the cells containing the protein which permits Changes in the constant region will, in general, be made in those cells to be readily identified. In sum, “expression vec order to improve the cellular process characteristics, such as tor” is given a functional definition, and any DNA sequence complement fixation, interaction with membranes, and other 10 which is capable of effecting expression of a specified con effector functions. Changes in the variable region will be tained DNA code is included in this term, as it is applied to the made in order to improve the antigen binding characteristics. specified sequence. As at present, such vectors are frequently The antibody can also be engineered so as to aid the specific in the form of plasmids, thus "plasmid' and “expression delivery of a toxic agent according to the “magic bullet” vector” are often used interchangeably. However, the inven concept. Alterations, can be made by Standard recombinant 15 tion is intended to include Such other forms of expression techniques and also by oligonucleotide-directed mutagenesis vectors which serve equivalent functions and which may, techniques (Dalbadie-McFarland, etal Proc. Natl. Acad. Sci. from time to time become known in the art. (USA), 79:6409 (1982)). "Recombinant host cells' refers to cells which have been “Univalent antibodies' refers to aggregations which com transformed with vectors constructed using recombinant prise a heavy chain/light chain dimer bound to the Fc (or DNA techniques. As defined herein, the antibody or modifi stem) region of a second heavy chain. Such antibodies are cation thereof produced by a recombinant host cell is by specific for antigen, but have the additional desirable property virtue of this transformation, rather than in such lesser of targeting tissues with specific antigenic Surfaces, without amounts, or more commonly, in Such less than detectable causing its antigenic effectiveness to be impaired—i.e., there amounts, as would be produced by the untransformed host. is no antigenic modulation. This phenomenon and the prop 25 In descriptions of processes for isolation of antibodies erty of univalent antibodies in this regard is set forth in Glen from recombinant hosts, the terms “cell' and “cell culture' nie, M.J., et al., Nature, 295: 712 (1982). Univalent antibod are used interchangeably to denote the Source of antibody ies have heretofore been formed by proteolysis. unless it is clearly specified otherwise. In other words, recov “Fab’ region refers to those portions of the chains which ery of antibody from the “cells' may mean either from spun are roughly equivalent, or analogous, to the sequences which 30 down whole cells, or from the cell culture containing both the comprise the Y branch portions of the heavy chain and to the medium and the Suspended cells. light chain in its entirety, and which collectively (in aggre gates) have been shown to exhibit antibody activity. “Fab B. Host Cell Cultures and Vectors protein', which protein is one of the aspects of the invention, includes aggregates of one heavy and one light chain (com 35 The vectors and methods disclosed herein are suitable for monly known as Fab'), as well as tetramers which correspond use in host cells over a wide range of prokaryotic and eukary to the two branch segments of the antibody Y. (commonly otic organisms. known as F(ab)a), whether any of the above are covalently or In general, of course, prokaryotes are preferred for cloning non-covalently aggregated, so long as the aggregation is of DNA sequences in constructing the vectors useful in the capable of selectively reacting with a particular antigen or 40 invention. For example, E. coli K12 strain 294 (ATCC No. antigen family. Fab antibodies have, as have univalent ones, 31446) is particularly useful. Other microbial strains which been formed heretofore by proteolysis, and share the property may be used include E. coli Strains such as E. coli B, and E. of not eliciting antigen modulation on target tissues. How coli X1776 (ATTC No. 31537). ever, as they lack the “effector Fc portion they cannot effect, These examples are, of course, intended to be illustrative for example, lysis of the target cell by macrophages. 45 rather than limiting. “Fab protein’ has similar subsets according to the defini Prokaryotes may also be used for expression. The afore tion of the present invention as does the general term “anti mentioned strains, as well as E. coli W3110 (F, , pro bodies' or “immunoglobulins’. Thus, “mammalian Fab pro totrophic, ATTCNo. 27325), bacillisuch as Bacillus subtilus, tein, “hybrid” Fab protein “chimeric' Fab and “altered” Fab and other enterobacteriaceae such as Salmonella typhimu protein are defined analogously to the corresponding defini 50 rium or Serratia marcesans, and various Pseudomonas spe tions set forth in the previous paragraphs for the various types cies may be used. of antibodies. In general, plasmid vectors containing replicon and control Individual heavy or light chains may of course be “mam sequences which are derived from species compatible with malian', 'chimeric' or "altered in accordance with the the host cell are used in connection with these hosts. The above. As will become apparent from the detailed description 55 vector ordinarily carries a replication site, as well as marking of the invention, it is possible, using the techniques disclosed sequences which are capable of providing phenotypic selec to prepare other combinations of the four-peptide chain tion in transformed cells. For example, E. coli is typically aggregates, besides those specifically defined. Such as hybrid transformed using pBR322, a plasmid derived from an E. coli antibodies containing chimeric light and mammalian heavy species (Bolivar, et al., Gene 2: 95 (1977)). pBR322 contains chains, hybrid Fab proteins containing chimeric Fab proteins 60 genes for ampicillin and tetracycline resistance and thus pro of heavy chains associated with mammalian light chains, and vides easy means for identifying transformed cells. The so forth. pBR322 plasmid, or other microbial plasmid must also con “Expression vector” includes vectors which are capable of tain, or be modified to contain, promoters which can be used expressing DNA sequences contained therein, i.e., the coding by the microbial organism for expression of its own proteins. sequences are operably linked to other sequences capable of 65 Those promoters most commonly used in recombinant DNA effecting their expression. It is implied, although not always construction include the B-lactamase (penicillinase) and lac explicitly stated, that these expression vectors must be repli tose promoter systems (Chang etal, Nature, 275: 615 (1978); US 7,923,221 B1 9 10 Itakura, et al, Science, 198: 1056 (1977); (Goeddel, et al particularly useful because both are obtained easily from the Nature 281: 544 (1979)) and a tryptophan (trp) promoter virus as a fragment which also contains the SV40 viral origin system (Goeddel, et al. Nucleic Acids Res., 8: 4057 (1980); of replication (Fiers, et al. Nature, 273: 113 (1978)) incorpo EPO Appl Publ No. 0036776). While these are the most rated herein by reference. Smaller or larger SV40 fragments commonly used, other microbial promoters have been dis may also be used, provided there is included the approxi covered and utilized, and details concerning their nucleotide mately 250 bp sequence extending from the Hind III site sequences have been published, enabling a skilled worker to toward the Bgl I site located in the viral origin of replication. ligate them functionally with plasmid vectors (Siebenlist, et Further, it is also possible, and often desirable, to utilize al, Cell 20: 269 (1980)). promoter or control sequences normally associated with the In addition to prokaryates, eukaryotic microbes, such as 10 desired gene sequence, provided Such control sequences are yeast cultures may also be used. Saccharomyces cerevisiae, compatible with the host cell systems. or common baker's yeast is the most commonly used among An origin of replication may be provided either by con eukaryotic microorganisms, although a number of other struction of the vector to include an exogenous origin, Such as strains are commonly available. For expression in Saccharo may be derived from SV40 or other viral (e.g. Polyoma, myces, the plasmid YRp7, for example, (Stinchcomb, et al. 15 Adeno, VSV, BPV, etc.) source, or may be provided by the Nature, 282:39 (1979); Kingsman etal, Gene, 7: 141 (1979); host cell chromosomal replication mechanism. If the vectoris Tschemper, et al. Gene, 10: 157 (1980)) is commonly used. integrated into the host cell chromosome, the latter is often This plasmid already contains the trp1 gene which provides a sufficient. selection marker for a mutant strain of yeast lacking the It will be understood that this invention, although described ability to grow in tryptophan, for example ATCC No. 44076 herein in terms of a preferred embodiment, should not be or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presence of construed as limited to those host cells, vectors and expres the trp 1 lesion as a characteristic of the yeast host cell genome sion systems exemplified. then provides an effective environment for detecting transfor mation by growth in the absence of tryptophan. C. Methods Employed Suitable promoting sequences in yeast vectors include the 25 promoters for 3-phosphoglycerate kinase (Hitzeman, et al., J. C.1 Transformation Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess, etal, J. Adv. Enzyme Reg., 7: 149 (1968); Holland, etal, If cells without formidable cell wall barriers are used as Biochemistry, 17: 4900 (1978)), such as enolase, glyceralde host cells, transfection is carried out by the calcium phosphate hyde-3-phosphate dehydrogenase, hexokinase, pyruvate 30 precipitation method as described by Graham and Vander Eb. decarboxylase, phosphofructokinase, glucose-6-phosphate Virology, 52: 546 (1978). However, other methods for intro isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tri ducing DNA into cells such as by nuclear injection or by osephosphate isomerase, phosphoglucose isomerase, and protoplast fusion may also be used. glucokinase. In constructing Suitable expression plasmids, If prokaryotic cells or cells which contain substantial cell the termination sequences associated with these genes are 35 wall constructions are used, the preferred method of transfec also ligated into the expression vector 3' of the sequence tion is calcium treatment using calcium chloride as described desired to be expressed to provide polyadenylation of the by Cohen, F. N. etal Proc. Natl. Acad. Sci. (USA), 69: 2110 mRNA and termination. Other promoters, which have the (1972). additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydroge 40 C.2 Vector Construction nase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the afore Construction of Suitable vectors containing the desired mentioned glyceraldehyde-3-phosphate dehydrogenase, and coding and control sequences employ standard ligation tech enzymes responsible for maltose and galactose utilization niques. Isolated plasmids or DNA fragments are cleaved, (Holland, ibid.). Any plasmid vector containing yeast-com 45 tailored, and religated in the form desired to form the plas patible promoter, origin of replication and termination mids required. The methods employed are not dependent on sequences is suitable. the DNA source, or intended host. In addition to microorganisms, cultures of cells derived Cleavage is performed by treating with restriction enzyme from multicellular organisms may also be used as hosts. In (or enyzmes) in Suitable buffer. In general, about 1 ugplasmid principle, any such cell culture is workable, whether from 50 or DNA fragments is used with about 1 unit of enzyme in vertebrate or invertebrate culture. However interest has been about 20 ul of buffer solution. (Appropriate buffers and sub greatest in Vertebrate cells, and propogation of Vertebrate strate amounts for particular restriction enzymes are specified cells in culture (tissue culture) has become a routine proce by the manufacturer.) Incubation times of about 1 hour at 37° dure in recent years (Tissue Culture, Academic Press, Kruse C. are workable. After incubations, protein is removed by and Patterson, editors (1973)). Examples of such useful host 55 extraction with phenol and chloroform, and the nucleic acid is cell lines are VERO and HeLa cells, Chinese hamster ovary recovered from the aqueous fraction by precipitation with (CHO) cell lines, and W138, BHK, COS-7 and MDCK cell ethanol. lines. Expression vectors for such cells ordinarily include (if If blunt ends are required, the preparation is treated for 15 necessary) an origin of replication, a promoter located in front minutes at 15° with 10 units of E. coli DNA Polymerase I of the gene to be expressed, along with any necessary ribo 60 (Klenow), phenol-chloroform extracted, and ethanol precipi Some binding sites, RNA splice sites, polyadenylation site, tated. and transcriptional terminator sequences. Size separation of the cleaved fragments is performed For use in mammalian cells, the control functions on the using 6 percent polyacrylamide gel described by Goeddel, D., expression vectors are often provided by viral material. For et al. Nucleic Acids Res., 8: 4057 (1980) incorporated herein example, commonly used promoters are derived from 65 by reference. polyoma, Adenovirus 2, and most frequently Simian Virus 40 For ligation, approximately equimolar amounts of the (SV40). The early and late promoters of SV40 virus are desired components, Suitably end tailored to provide correct US 7,923,221 B1 11 12 matching are treated with about 10 units T4 DNA ligase per The desired gene fragments are excised and tailored to 0.5ug DNA. (When cleaved vectors are used as components, assure appropriate reading frame with the control segments it may be useful to prevent religation of the cleaved vector by when inserted into Suitable expression vectors. Typically, pretreatment with bacterial alkaline phosphatase.) nucleotides are added to the 5' end to include a start signal and In the examples described below correct ligations for plas a Suitably positioned restriction endonuclease site. mid construction are confirmed by transforming E. coli K12 The tailored gene sequence is then positioned in a vector strain 294 (ATCC 31446) with the ligation mixture. Success which contains a promoter in reading frame with the gene and ful transformants were selected by amplicillin or tetracycline compatible with the proposed host cell. A number of plasmids resistance depending on the mode of plasmid construction. such as those described in U.S. Pat. Nos. 307,473; 291,892; Plasmids from the transformants were then prepared, ana 10 and 305,657, have been described which already contain the lyzed by restriction and/or sequenced by the method of Mess appropriate promoters, control sequences, ribosome binding ing, et al. Nucleic Acids Res., 9:309 (1981) or by the method sites, and transcription termination sites, as well as conve of Maxam, et al. Methods in Enzymology, 65:499 (1980). nient markers. In the present invention, the gene coding for the light chain D. Outline of Procedures 15 and that coding for the heavy chain are recovered separately by the procedures outlined above. Thus they may be inserted D. 1 Mammalian Antibodies into separate expression plasmids, or together in the same plasmid, so long as each is under Suitable promoter and trans The first type of antibody which forms a part of this inven lation control. tion, and is prepared by the methods thereof, is “mammalian The expression vectors constructed above are then used to antibody’-one wherein the heavy and light chains mimic the transform suitable cells. The light and heavy chains may be amino acid sequences of an antibody otherwise produced by transformed into separate cell cultures, either of the same or a mature mammalian B lymphocyte either in situ or when of differing species; separate plasmids for light and heavy fused with an immortalized cell as part of a hybridoma cul chain may be used to co-transform a single cell culture, or, ture. In outline, these antibodies are produced as follows: 25 finally, a single expression plasmid containing both genes and Messenger RNA coding for heavy or light chain is isolated capable of expressing the genes for both light and heavy chain from a suitable source, either mature B cells or a hybridoma may be transformed into a single cell culture. culture, employing standard techniques of RNA isolation, Regardless of which of the three foregoing options is cho and the use of oligo-dT cellulose chromatography to segre sen, the cells are grown under conditions appropriate to the gate the poly-A mRNA. The poly-A mRNA may, further, be 30 production of the desired protein. Such conditions are prima fractionated to obtain sequences of Sufficient size to code for rily mandated by the type of promoter and control systems the amino acid sequences in the light or heavy chain of the used in the expression vector, rather than by the nature of the desired antibody as the case may be. desired protein. The protein thus produced is then recovered A cDNA library is then prepared from the mixture of from the cell culture by methods known in the art, but choice mRNA using a suitable primer, preferably a nucleic acid 35 of which is necessarily dependent on the form in which the sequence which is characteristic of the desired cDNA. Such a protein is expressed. For example, it is common for mature primer may be hypothesized and synthesized based on the heterologous proteins expressed in E. coli to be deposited amino acid sequence of the antibody if the sequence is known. within the cells as insoluble particles which require cell lysis In the alternative cDNA from unfractionated poly-A mRNA and solubilization in denaturant to permit recovery. On the from a cell line producing the desired antibody or poly-dT 40 other hand, proteins under proper synthesis circumstances, in may also be used. The resulting cDNA is optionally size yeast and bacterial strains, can be secreted into the medium fractionated on polyacrylamide gel and then extended with, (yeast and gram positive bacteria) or into the periplasmic for example, dC residues for annealing with pBR322 or other space (gram negative bacteria) allowing recovery by less suitable cloning vector which has been cleaved by a suitable drastic procedures. Tissue culture cells as hosts also appear, in restriction enzyme, such as Pst I, and extended with dG resi 45 general, to permit reasonably facile recovery of heterologous dues. Alternative means of forming cloning vectors contain proteins. ing the cDNA using other tails and other cloning vector When heavy and light chain are coexpressed in the same remainder may, of course, also be used but the foregoing is a host, the isolation procedure is designed so as to recover standard and preferable choice. A suitable host cell strain, reconstituted antibody. This can be accomplished in vitro as typically E. coli, is transformed with the annealed cloning 50 described below, or might be possible in vivo in a microor vectors, and the Successful transformants identified by means ganism which secretes the IgG chains out of the reducing of for example, tetracycline resistance or other phenotypic environment of the cytoplasm. A more detailed description is characteristic residing on the cloning vector plasmid. given in D.2, below. Successful transformants are picked and transferred to microtiter dishes or other support for further growth and 55 D.2 Chain Recombination Techniques preservation. Nitrocellulose filter imprints of these growing cultures are then probed with suitable nucleotide sequences The ability of the method of the invention to produce heavy containing bases known to be complementary to desired and light chains or portions thereof, in isolation from each sequences in the cDNA. Several types of probe may be used, other offers the opportunity to obtain unique and unprec preferably synthetic single stranded DNA sequences labeled 60 edented assemblies of immunoglobulins, Fab regions, and by kinasing with ATP. The cells fixed to the nitrocellulose univalent antibodies. Such preparations require the use of filter are lysed, the DNA denatured, and then fixed before techniques to reassemble isolated chains. Such means are reaction with kinased probe. Clones which successfully known in the art, and it is, thus, appropriate to review them hybridize are detected by contact with a photoplate, then here. plasmids from the growing colonies isolated and sequenced 65 While single chain disulfide bond containing proteins have by means known in the art to verify that the desired portions been reduced and reoxidized to regenerate in highyield native of the gene are present. structure and activity (Freedman, R. B., et al. In Enzymology US 7,923,221 B1 13 14 of Post Translational Modification of Proteins, I: 157-212 contaminated, molecules. The variations also inhere in the (1980) Academic Press, NY.), proteins which consist of dis ability to isolate production of individual chains, and thus continuous polypeptide chains held together by disulfide create novel assemblies. bonds are more difficult to reconstruct in vitro after reductive Briefly, since genetic manipulations permit reconstruction cleavage. Insulin, a cameo case, has received much experi of genomic material in the process of construction of expres mental attention over the years, and can now be reconstructed sion vectors, such reconstruction can be manipulated to pro so efficiently that an industrial process has been built around duce new coding sequences for the components of “natural it (Chance, R. E., et al. In Peptides. Proceedings of the antibodies or immunoglobulins. As discussed in further detail Seventh Annual American Peptide Symposium (Rich, D. H. below, the coding sequence for a mammalian heavy chain and Gross, E., eds.)721-728, Pierce Chemical Co., Rockford, 10 may not be derived entirely from a single source or single Ill. (1981)). species, but portions of a sequence can be recovered by the Immunoglobulin has proved a more difficult problem than techniques described in D.1 from differing pools of mRNA, insulin. The tetramer is stabilized intra and intermolecularly Such as murine-murine hybridomas, human-murine hybrido by, 15 or more disulfide bonds. It has been possible to recom mas, or B cells differentiated in response to a series of antigen bine heavy and light chains, disrupted by cleavage of only the 15 challenges. The desired portions of the sequences in each case interchain disulfides, to regain antibody activity even without can be recovered using the probe and analysis techniques restoration of the inter-chain disulfides (Edelman, G. M., et described in D.1, and recombined in an expression vector al., Proc. Natl. Acad. Sci. (USA) 50: 753 (1963)). In addition, using the same ligation procedures as would be employed for active fragments of IgG formed by proteolysis (Fab frag portions of the same model sequence. Such chimeric chains ments of -50,000 MW) can be split into their fully reduced can be constructed of any desired length; hence, for example, heavy chain and light chain components and fairly efficiently a complete heavy chain can be constructed, or only sequence reconstructed to give active antibody (Haber, E., Proc. Natl. for the Fab region thereof. Acad. Sci. (USA) 52: 1099 (1964); Whitney, P. L., et al., Proc. The additional area of flexibility which arises from the use Natl. Acad. Sci. (USA) 53: 524 (1965)). Attempts to reconsti 25 of recombinant techniques results from the power to produce tute active antibody from fully reduced native IgG have been heavy and light chains or fragments thereof in separate cul largely unsuccessful, presumably due to insolubility of the tures or of unique combinations of heavy and light chain in reduced chains and of side products or intermediates in the the same culture, and to prevent reconstitution of the antibody refolding pathway (see discussion in Freedman, M. H., et al., or immunoglobulin aggregation until the Suitable compo J. Biol. Chem. 241: 5225 (1966)). If, however, the immuno 30 nents are assembled. Thus, while normal antibody production globulin is randomly modified by polyalanylation of its results automatically in the formation of “mammalian anti lysines before complete reduction, the separated chains have bodies' because the light and heavy chain portions are con the ability to recover antigen-combining activity upon reoxi structed in response to a particular determinant in the same dation (ibid). A particularly Suitable method for immunoglobulin recon 35 cell, the methods of the present invention present the oppor stitution is derivable from the now classical insulin recombi tunity to assemble entirely new mixtures. Somewhat limited nation studies, wherein starting material was prepared by quantities of “hybrid antibodies have been produced by oxidative sulfitolysis, thus generating thiol-labile S-sulfonate “quadromas’ i.e., fusions of two hybridoma cell cultures groups at all cysteines in the protein, non-reductively break which permit random assemblies of the heavy and light ing disulfides (Chance et al. (supra)). Oxidative sulfitolysis is 40 chains so produced. a mild disulfide cleavage reaction (Means, G. E., et al., The present invention permits a more controlled assembly Chemical Modification of Proteins, Holden-Day, San Fran of desired chains, either by mixing the desired chains in vitro, cisco (1971)) which is sometimes more gentle than reduction or by transforming the same culture with the coding (Wetzel, R., Biochemistry, submitted (1983)), and which gen sequences for the desired chains. erates derivatives which are stable until exposed to mild 45 reducing agent at which time disulfide reformation can occur D.4 Composite Immunoglobulins via thiol-disulfide interchange (Morehead, H., et al. Biochem istry, in press, (1983)). In the present invention the heavy and The foregoing procedure, which describes in detail the light chain S-Sulfonates generated by oxidative Sulfitolysis recombinant production of mammalian antibodies is were reconstituted utilizing both air oxidation and thiol-dis 50 employed with Some modifications to construct the remain ulfide interchange to drive disulfide bond formation. The ing types of antibodies or NSIs encompassed by the present general procedure is set forth in detail in U.S. Pat. No. 452, invention. To prepare the particular embodiment of compos 187, filed Dec. 22, 1982, incorporated herein by reference. ite non-specific immunoglobulin wherein the homology of the chains corresponds to the sequences of immunoglobulins D.3 Variants Permitted by Recombinant Technology 55 of different specificities, it is of course, only necessary to prepare the heavy and light chains in separate cultures and Using the techniques described in paragraphs D.1 and D.2, reassemble them as desired. additional operations which were utilized to gain efficient For example, in order to make an anti-CEA light chain/ production of mammalian antibody can be varied in quite anti-hepatitis heavy chain composite antibody, a Suitable straightforward and simple ways to produce a great variety of 60 source for the mRNA used as a template for the light chain modifications of this basic antibody form. These variations clone would comprise, for instance, the anti CEA producing are inherent in the use of recombinant technology, which cell line of paragraph E.1. The mRNA corresponding to heavy permits modification at a genetic level of amino acid chain would be derived from B cells raised in response to sequences in normally encountered mammalian immunoglo hepatitis infection or from hybridomain which the B cell was bulin chains, and the great power of this approach lies in its 65 of this origin. It is clear that Such composites can be ability to achieve these variations, as well as in its potential for assembled using the methods of the invention almost at will, economic and specific production of desired scarce, and often and are limited only by available sources of mRNA suitable US 7,923,221 B1 15 16 for use as templates for the respective chains. All other fea bodies, be used to target antigenic Surfaces of tissues, such as tures of the process are similar to those described above. tumors, but, unlike ordinary antibodies, they do not cause the antigenic Surfaces of the target tissue to retreat and become D.5 Hybrid Antibodies non-receptive. Ordinary antibody use results in aggregation and Subsequentinactivation, for several hours, of such surface Hybrid antibodies are particularly useful as they are antigens. capable of simultaneous reaction with more than one antigen. The method of construction of univalent antibodies is a Pairs of heavy and light chains corresponding to chains of straightforward application of the invention. The gene for antibodies for different antigens, such as those set forth in heavy chain of the desired Fc region is cleaved by restriction paragraph D.4 are prepared in four separate cultures, thus 10 enzymes, and only that portion coding for the desired Fc preventing premature assembly of the tetramer. Subsequent region expressed. This portion is then bound using the tech mixing of the four separately prepared peptides then permits nique of D.2 to separately produced heavy chain the desired assembly into the desired tetramers. While random aggrega pairs separated from heavy/heavy and Fc/Fc combinations, tion may lead to the formation of considerable undesired and separately produced light chain added. Pre-binding of the product, that portion of the product in which homologous 15 two heavy chain portions thus diminishes the probability of light and heavy chains are bound to each other and mis formation of ordinary antibody. matched to another pair gives the desired hybrid antibody. D.9 Fab Protein D.6 Chimeric Antibodies Similarly, it is not necessary to include the entire gene for For construction of chimeric antibodies (wherein, for the heavy chain portion. All of the aforementioned variations example, the variable sequences are separately derived from can be Superimposed on a procedure for Fab protein produc the constant sequences) the procedures of paragraph D.1 and tion and the overall procedure differs only in that that portion D.2 are again applicable with appropriate additions and modi of the heavy chain coding for the amino terminal 220 amino fications. A preferred procedure is to recover desired portions 25 acids is employed in the appropriate expression vector. of the genes encoding for parts of the heavy and light chains from Suitable, differing, Sources and then to religate these E. Specific Examples of Preferred Embodiments fragments using restriction endonucleases to reconstruct the gene coding for each chain. The invention has been described above in general terms For example, in a particularly preferred chimeric construc 30 and there follow several specific examples of embodiments tion, portions of the heavy chain gene and of the light chain which set forth details of experimental procedure in produc gene which encode the variable sequences of antibodies pro ing the desired antibodies. Example E.1 sets forth the general duced by a murine hybridoma culture are recovered and procedure for preparing anti CEA antibody components, i.e. cloned from this culture and gene fragments encoding the for a “mammalian antibody’. Example E.3 sets forth the constant regions of the heavy and light chains for human 35 procedure for reconstitution and thus is applicable to prepa antibodies recovered and cloned from, for example, human ration of mammalian, composite, hybrid and chimeric immu myeloma cells. Suitable restriction enzymes may then be noglobulins, and Fab proteins and univalent antibodies. used to ligate the variable portions of the mouse gene to the Example E.4 sets forth the procedure for tailoring the heavy constant regions of the human gene for each of the two chains. or light chain so that the variable and constant regions may be The chimeric chains are produced as set forth in D. 1, aggre 40 derived from different sources. Example E.5 sets forth the gated as set forth in D.2 and used in the same manner as the method of obtaining a shortened heavy chain genome which non-chimeric forms. Of course, any splice point in the chains permits the production of the Fab regions and, in an analogous can be chosen. manner, Fc region. The examples set forth below are included for illustrative D.7 Altered Antibodies 45 purposes and do not limit the scope of the invention. E.1 Construction of Expression Vectors for Murine Anti Altered antibodies present, in essence, an extension of CEA Antibody Chains and Peptide Synthesis chimeric ones. Again, the techniques of D.1 and D.2 are Carcinoembryonic antigen (CEA) is associated with the applicable; however, rather than splicing portions of the Surface of certain tumor cells of human origin (Gold, P. et al., chain(s), Suitable amino acid alterations, deletions or addi 50 J. Exp. Med., 122: 467 (1965)). Antibodies which bind to tions are made using available techniques such as mutagen CEA (anti-CEA antibodies) are useful in early detection of esis (Supra). For example, genes which encode antibodies these tumors (Van Nagell, T. R., et al., Cancer Res. 40: 502 having diminished complement fixation properties, or which (1980)), and have the potential for use in treatment of those have enhanced metal binding capacities are prepared using human tumors which appear to support CEA at their Surfaces. Such techniques. The latter type may, for example, take 55 A mouse hybridoma cell line which secretes anti-CEA anti advantage of the known gene sequence encoding metalothio bodies of the Igy class, CEA.66-E3, has been prepared as nein II (Karin, M., et al., Nature, 299: 797 (1982)). The described by Wagener, C, et al., J. Immunol. (in press) which chelating properties of this molecular fragment are useful in is incorporated herein by reference, and was used as mRNA carrying heavy metals to tumor sites as an aid in tumor imag source. The production of anti CEA antibodies by this cell ing (Scheinberg, D. A., et al., Science, 215: 19 (1982). 60 line was determined. The N-terminal sequences of the anti bodies produced by these cells was compared with those of D.8 Univalent Antibodies monoclonal anti CEA as follows. Purified IgG was treated with PCAse (Podell, D.N., et al., BBRC 81: 176 (1978)), and In another preferred embodiment, antibodies are formed then dissociated in 6M guanidine hydrochloride, 10 mM which comprise one heavy and light chain pair coupled with 65 2-mercaptoethanol (1.0 mg of immunoglobulin, 5 min, 100° the Fc region of a third (heavy) chain. These antibodies have C. water bath). The dissociated chains were separated on a a particularly useful property. They can, like ordinary anti Waters Associates alkyl phenyl column using a linear gradi US 7,923,221 B1 17 18 ent from 100 percent A (0.1 percent TFA-water) to 90 percent Both probes were synthesized by the phosphotriester B (TFA/HO/MeCN 0.1/9.9/90) at a flow rate of 0.8 ml/min. method described in German Offenlegungschrift 2644432. Three major peaks were eluted and analyzed on SOS gels by incorporated herein by reference, and made radioactive by silver staining. The first two peaks were purelight chain (MW kinasing as follows: 250 ng of deoxyoligonucleotide were 25,000 daltons), the third peak showed a (7:3) mixture of 5 combined in 25ul of 60 mM Tris HCl (pH8), 10 mM MgCl, heavy and light chain. 1.2 nmoles of light chain were 15 mM beta-mercaptoethanol, and 100 uCi (y-'P)ATP (Am sequenced by the method of Shively, J. E., Methods in Enzy ersham, 5000 Ci/mMole).5 units of T4 polynucleotide kinase mology, 79: 31 (1981), with an NH-terminal yield of 0.4 were added and the reaction was allowed to proceed at 37°C. nmoles. A mixture of heavy and light chains (3 nmoles) was for 30 minutes and terminated by addition of EDTA to 20 10 mM. also sequenced, and sequence of light chain was deducted E.1.4 Screening of Colony Library for Kappa or Gamma from the double sequence to yield the sequence of the heavy Chain Sequences chain. ~2000 colonies prepared as described in paragraph E.1.2 In the description which follows, isolation and expression were individually inoculated into wells of microtitre dishes of the genes for the heavy and light chains for anti CEA 15 containing LB (Miller, Experiments in Molecular Genetics, antibody produced by CEA.66-E3 are described. As the con p. 431-3, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. stant regions of these chains belong to the gamma and kappa (1972))+5ug/ml tetracycline and stored at -20°C. after addi families, respectively, “light chain' and "kappa chain', and tion of DMSO to 7 percent. Individual colonies from this "heavy chain” and “gamma chain', respectively, are used library were transferred to duplicate sets of Schleicher and interchangeably below. Schuell BA85/20 nitrocellulose filters and grown on agar E.1.1 Isolation of Messenger RNA for Anti CEA Light and plates containing LB+5ug/ml tetracycline. After ~10 hours Heavy (Kappa and Gamma) Chains growth at 37° C. the colony filters were transferred to agar Total RNA from CEA.66-E3 cells was extracted essen plates containing LB+5 ug/ml tetracycline and 12.5 ug/ml tially as reported by Lynch et al. Virology, 98: 251 (1979). chloramphenicol and reincubated overnight at 37° C. The Cells were pelleted by centrifugation and approximately 1g 25 DNA from each colony was then denatured and fixed to the portions of pellet resuspended in 10 ml of 10 mM NaCl, 10 filter by a modification of the Grunstein-Hogness procedure mM Tris HCl (pH 7.4), 1.5 mMMgCl. The resuspended cells as described in Grunstein et al., Proc. Natl. Acad. Sci. (USA) were lysed by addition of non-ionic detergent NP-40 to a final 72: 3961 (1975), incorporated herein by reference. Each filter concentration of 1 percent, and nuclei removed by centrifu was floated for 3 minutes on 0.5 NNaOH, 1.5M NaCl to lyse gation. After addition of SDS (pH 7.4) to 1 percent final 30 the colonies and denature the DNA then neutralized by float concentration, the Supernatant was extracted twice with 3 ml ing for 15 minutes on 3 MNaCl, 0.5M Tris HCl (pH 7.5). The portions of phenol (redistilled)/chloroform:isoamyl alcohol filters were then floated for an additional 15 minutes on 25:1 at 4°C. The aqueous phase was made 0.2 Min NaCl and 2xSSC, and subsequently baked for 2 hours in an 80° C. total RNA was precipitated by addition of two volumes of 100 vacuum oven. The filters were prehybridized for -2 hours at percent ethanol and overnight storage at -20°C. After cen 35 room temperature in 0.9 MNaCl, 1xDenhardts, 100 mM Tris trifugation, polyA mRNA was purified from total RNA by HCl (pH 7.5), 5 mM Na-EDTA, 1 mM ATP, 1 M sodium oligo-dT cellulose chromatography as described by Aviv and phosphate (dibasic), 1 mM sodium pyrophosphate, 0.5 per Leder, Proc. Natl. Acad. Sci. (USA), 69: 1408 (1972). 142 ug cent NP-40, and 200 ug/ml E. coli t-RNA, and hybridized in of polyA mRNA was obtained from 1 g cells. the same solution overnight, essentially as described by Wal E.1.2 Preparation of E. coli Colony Library Containing 40 lace et al. Nucleic Acids Research 9: 879 (1981) using ~40x Plasmids with Heavy and Light DNA Sequence Inserts 10 cpm of either the kinased kappa or gamma probe 5ug of the unfractionated polyA mRNA prepared in para described above. graph E.1.1 was used as template for oligo-dT primed prepa After extensive washing at 37°C. in 6xSSC, 0.1 percent ration of double-stranded (ds) cDNA by standard procedures SDS, the filters were exposed to Kodak XR-5 X-ray film with as described by Goeddelet al., Nature 281: 544 (1979) and 45 DuPont Lightning-Plus intensifying screens for 16-24 hours Wickens et al., J. Biol. Chem. 253: 2483 (1978) incorporated at -80°C. Approximately 20 colonies which hybridized with herein by reference. The cDNA was size fractionated by 6 kappa chain probe and 20 which hybridized with gamma percent polyacrylamide gel electrophoresis and 124 ng of ds chain probe were characterized. cDNA greater than 600 base pairs in length was recovered by E.1.5 Characterization of Colonies which Hybridize to electroelution. A 20 ng portion of ds cDNA was extended 50 Kappa DNA Sequence Probe with deoxy Cresidues using terminal deoxynucleotidyltrans Plasmid DNAs isolated from several different transfor ferase as described in Chang et al., Nature 275: 617 (1978) mants which hybridized to kappa chain probe were cleaved incorporated herein by reference, and annealed with 200ng of with Pst I and fractionated by polyacrylamide gel electro the plasmidpBR322 (Bolivaret al., Gene 2: 95 (1977)) which phoresis (PAGE). This analysis demonstrated that a number had been cleaved with Pst I and tailed with deoxy G. Each 55 of plasmid DNAs contained cDNA inserts large enough to annealed mixture was then transformed into E. coli K12 strain encode full length kappa chain. The complete nucleotide 294 (ATCC No. 31446). Approximately 8500ampicillinsen sequence of the cDNA insert of one of these plasmids was sitive, tetracycline resistant transformants were obtained. determined by the dideoxynucleotide chain termination E.1.3 Preparation of Synthetic Probes method as described by Smith, Methods Enzymol. 65, 560 The 14mer, 5' GGTGGGAAGATGGA 3' complementary 60 (1980) incorporated herein by reference after subcloning to the coding sequence of constant region for mouse restriction endonuclease cleavage fragments into M13 Vec MOPC21 kappa chain which begins 25 basepairs 3' of the tors (Messing et al., Nucleic Acids Research 9: 309 (1981). variable region DNA sequence was used as kappa chain FIG. 2 shows the nucleotide sequence of the cDNA insert of probe. A 15mer, 5' GACCAGGCATCCCAG3', complemen pK17G4 and FIG.3 shows the gene sequence with the corre tary to a coding sequence located 72 basepairs 3' of the 65 sponding amino acid sequence. Thus, the entire coding region variable region DNA sequence for mouse MOPC21 gamma of mouse anti-CEA kappa chain was isolated on this one large chain was used to probe gamma chain gene. DNA fragment. The amino acid sequence of kappa chain, US 7,923,221 B1 19 20 deduced from the nucleotide sequence of the pK17G4 cDNA the trp promoter, flanked by EcoRI sites and was used to insert, corresponds perfectly with the first 23 N-terminal prepare pHGH207-1. pHGH207 was digested with BamH 1, amino acids of mature mouse anti-CEA kappa chain as deter followed by partial digestion with EcoRI. The largest frag mined by amino acid sequence analysis of purified mouse ment, which contains the entire trp promoter, was isolated and anti-CEA kappa chain. The coding region of pK17G4 con ligated to the largest EcoRI-BamHI fragment from pBR322, tains 27 basepairs or 9 amino acids of the presequence and and the ligation mixture used to transform E. coli 294. Tet' 642 basepairs or 214 amino acids of the mature protein. The Amp' colonies were isolated, and most of them contained mature unglycosylated protein (MW 24,553) has a variable pHGH207-1. pHGH207-1, which lacks the EcoR1 site region of 119 amino acids, including the J1 joining region of between the amp' gene and the trp promoter, was obtained by 12 amino acids, and a constant region of 107 amino acids. 10 partial digestion of pHGH207-1 with EcoRI, filling in the After the stop codon behind amino acid 215 begins 212 base pairs of 3' untranslated sequence up to the polyA addition. ends with Klenow and dNTPs, and religation. The kappa chain probe used to identify pK17G4 hybridizes to 5ug of pHGH207-1 was digested with EcoRI, and the nucleotides 374-388 (FIG. 2). ends extended to blunt ends using 12 units of DNA Poly E.1.6 Characterization of Colonies which Hybridize to 15 merase I in a 50 ul reaction containing 60 mM NaCl, 7 mM Gamma 1 DNA Probe MgCl, 7 mM Tris HCl (pH 7.4) and 1 mM in each dNTP at Plasmid DNA isolated from several transformants positive 37° C. for 1 hour, followed by extraction with phenol/CHCl for hybridization with the heavy chain gamma 1 probe was and precipitation with ethanol. The precipitated DNA was Subjected to Pst I restriction endonuclease analysis as digested with BamH I, and the large vector fragment (frag described in E.1.5. Plasmid DNAs demonstrating the largest ment 1) purified using 5 percent polyacrylamide gel electro cDNA insert fragments were selected for further study. phoresis, electroelution, phenol/CHCl extraction and etha Nucleotide sequence coding for mouse heavy (gamma-1) nol precipitation. chain, shows an Nicol restriction endonuclease cleavage site The DNA was resuspended in 50 ul of 10 mM Tris pH 8, 1 near the junction between variable and constant region. mM EDTA and treated with 500 units Bacterial Alkaline Selected plasmid DNAs were digested with both PstI and 25 Phosphatase (BAP) for 30' at 65° followed by phenol/CHCl NcoI and sized on polyacrylamide. This analysis allowed extraction and ethanol precipitation. identification of a number of plasmid DNAs that contain NcoI A DNA fragment containing part of the light chain restriction endonuclease sites, although none that demon sequence was prepared as follows: 7 g of pK17G4 DNA was strate cDNA insert fragments large enough to encode the digested with Pst I and the kappa chain containing cDNA entire coding region of mouse anti-CEA heavy chain. 30 insert was isolated by 6 percent gel electrophoresis, and elec In one plasmid isolated, p Y298 the cDNA insert of about 1300 bp contains sequence information for the 5' untranslated troelution. After phenol/CHCl extraction, ethanol precipita region, the signal sequence and the N-terminal portion of tion and resuspension in water, this fragment was digested heavy chain. Because py298 did not encode the C-terminal with Ava II. The 333 bp Pst I-Ava II DNA fragment was sequence for mouse anti-CEA gamma 1 chain, plasmid DNA 35 isolated and purified from a 6 percent polyacrylamide gel. was isolated from other colonies and screened with Pst and A 15 nucleotide DNA primer was synthesized by the phos NcoI. The C-terminal region of the cDNA insert of py11 was photriester method G. O. 2,644,432 (supra) and has the fol sequenced and shown to contain the stop codon, 3' untrans lowing sequence: lated sequence and that portion of the coding sequence miss ing from p Y298. 40 FIG. 4 presents the entire nucleotide sequence of mouse Met Asp Ile Val Met anti-CEA heavy chain (as determined by the dideoxynucle s' ATG GAC ATT GTT ATG 3." otide chain termination method of Smith, Methods Enzymol., The 5' methionine serves as the initiation codon. 500 ng of 65: 560 (1980)) and FIG. 5 includes the translated sequence. this primer was phosphorylated at the 5' end with 10 units T4 The amino acid sequence of gamma 1 (heavy chain) 45 DNA kinase in 20 ul reaction containing 0.5 mM ATP. -200 deduced from the nucleotide sequence of the pY298 cDNA ng of the Pst I-Ava II DNA fragment was mixed with the 2011 insert corresponds perfectly to the first 23 N-terminal amino of the phosphorylated primer, heated to 95°C. for 3 minutes acids of mature mouse anti-CEA gamma 1 chain as deter and quick frozen in a dry-ice ethanol bath. The denatured mined by amino acid sequence analysis of purified mouse DNA solution was made 60 mM NaCl, 7 mM MgCl, 7 mM anti-CEA gamma-1 chain. The coding region consists of 57 50 Tris HCl (pH 7.4), 12 mM in each dNTP and 12 units DNA basepairs or 19 amino acids of presequences and 1346 base Polymerase I-Large Fragment was added. After 2 hours incu pairs or 447 amino acids of mature protein. The mature ung bation at 37°C. this primer repair reaction was phenol/CHCl lycosolated protein (MW 52.258) has a variable region of 135 extracted, ethanol precipitated, and digested to completion amino acids, including a Dregion of 12 amino acids, and a J4 with Sau 3A. The reaction mixture was then electrophoresed joining region of 13 amino acids. The constant region is 324 55 on a 6 percent polyacrylamide gel and ~50 ng of the 182 amino acids. After the stop codon behind amino acid 447 basepair amino-terminal blunt-end to Sau3A fragment (frag begins 96 bp of 3' untranslated sequences up to the polyA ment 2) was obtained after electroelution. addition. The probe used to identify py298 and py11 hybrid 100 ng of fragment 1 (Supra) and 50 ng of fragment 2 were ized to nucleotides 528-542 (FIG. 4). combined in 20 ul of 20 mM Tris HCl (pH 7.5), 10 mM E. 1.7 Construction of a Plasmid for Direct Expression of 60 MgCl, 10 mM DTT, 2.5 mM ATP and 1 unit of 14 DNA Mouse Mature Anti-CEA Kappa Chain Gene, pKCE ligase. After overnight ligation at 14° C. the reaction was Atrp207-1* transformed into E. coli K12 strain 294. Restriction endonu FIG. 6 illustrates the construction of pKCEAtrp207-1* clease digestion of plasmid DNA from a number of amplicillin First, an intermediate plasmid pHGH207-1, having a resistant transformants indicated the proper construction and single trp promoter, was prepared as follows: 65 DNA sequence analysis proved the desired nucleotide The plasmid pHGH 207 (described in U.S. Pat. No. 307, sequence through the initiation codon of this new plasmid, 473, filed Oct. 1, 1981) has a double lac promoter followed by pKCEAInt1 (FIG. 6). US 7,923,221 B1 21 22 The remainder of the coding sequence of the kappa light I digestion. The 375 basepair blunt ended Ava II to Taq I chain gene was prepared as follows: fragment (fragment B) was isolated and purified by gel elec The Pst I cDNA insert fragment from 7 ug of K17G4 DNA trophoresis and electroelution. was partially digested with Ava II and the Ava II cohesive ends 9 ug of py298 was digested with Taq I and BamH I for were extended to blunt ends in a DNA Polymerase I large 5 isolation of the 496 basepair fragment (fragment C). fragment reaction. Following 6 percent polyacrylamide gel Approximately equimolar amounts of fragments A, B, and electrophoresis the 686 basepair Pst I to blunt ended Ava II C were ligated overnight at 14° in 20 ul reaction mixture, then DNA fragment was isolated, purified and subjected to HpaII transformed into E. coli strain 294. The plasmid DNA from restriction endonuclease digestion. The 497 basepair HpaII six ampicillin resistant transformants was committed to to blunt ended Ava II DNA fragment (fragment 3) was iso 10 lated and purified after gel electrophoresis. restriction endonuclease analysis and one plasmid DNA, 10 ug of pKCEAInt1 DNA was digested with Ava I, named pyCEAInt, demonstrated the correct construction of extended with DNA polymerase I large fragment, and the C-terminal portion of gamma 1 (FIG. 5). digested with Xba I. Both the large blunt ended Ava I to Xba To obtain the N-terminal sequences, 30 lug of py298 was I vector fragment and the small blunt ended Ava I to Xba I 15 digested with Pst I and the 628 basepair DNA fragment fragment were isolated and purified from a 6 percent poly encoding the N-terminal region of mouse anti-CEA gamma acrylamide gel after electrophoresis. The large vector frag chain was isolated and purified. This fragment was further ment (fragment 4) was treated with Bacterial Alkaline Phos digested with Alu I and Rsa I for isolation of the 280 basepair phatase (BAP), and the small fragment was digested with Hpa fragment. A 15 nucleotide DNA primer II, electrophoresed on a 6 percent polyacrylamide and the 169 basepair Xba I-HpaII DNA fragment (fragment 5) was puri fied. -75 ng of fragment 4, -50 ng of fragment 3 and ~50 ng met glu vall met lieu offragment 5 were combined in a T4DNA ligase reaction and 5. ATG GAA GTG ATG CTG 3." incubated overnight at 14°, and the reaction mixture trans was synthesized by the phosphotriester method (Supra). formed into E. coli K12 strain 294. Plasmid DNA from six 25 The 5' methionine serves as the initiation codon. 500 ng of ampicillin resistant transformants were analyzed by restric this synthetic oligomer primer was phosphorylated at the 5' tion endonuclease digestion. One plasmid DNA demon end in a reaction with 10 units T4DNA kinase containing 0.5 strated the proper construction and was designated mM ATP in 20 ul reaction mixture. -500 ng of the 280 pKCEAInt2. basepair Alu I-Rsa I DNA fragment was mixed with the Final construction was effected by ligating the K-CEA 30 phosphorylated primer. The mixture was heat denatured for 3 fragment, including the trp promoter from pKCEAInt2 into minutes at 95° and quenched in dry-ice ethanol. The dena pBR322(XAP). (pBR322(XAP) is prepared as described in tured DNA solution was made 60 mMNaCl, 7 mMMgCl, 7 U.S. Pat. No. 452,227, filed Dec. 22, 1982.) mM Tris HCl (pH 7.4), 12 mM in each dNTP and 12 units The K-CEA fragment was prepared by treating DNA Polymerase I-Large Fragment was added. After 2 hours pKCEAInt2 with Ava I, bluntending with DNA polymerase I 35 incubation at 37°C., this primer repair reaction was phenol/ (Klenow fragment) in the presence of DNTPs, digestion with CHCl extracted, ethanol precipitated, and digested to Pst I and isolation of the desired fragment by gel electro completion with HpaII. ~50 ng of the expected 125 basepair phoresis and electroelution. blunt-end to HpaII DNA fragment (fragment D) was purified The large vector fragment from pBR322(XAP) was pre from the gel. pared by successive treatment with EcoRI, bluntending with 40 A second aliquot of py298 DNA was digested with Pst I, the polymerase, and redigestion with Pst I, followed by isolation 628 basepair DNA fragment purified by polyacrylamide gel of the large vector fragment by electrophoresis and electro electrophoresis, and further digested with BamHI and HpaII. elution. The resulting 380 basepair fragment (fragment E) was puri The K-CEA and large vector fragments as prepared in the fied by gel electrophoresis. preceding paragraphs were ligated with T4 DNA ligase, and 45 ~5ug of pyCEAIntI was digested with EcoRI, the cohesive the ligation mixture transformed into E. coli as above. Plas ends were made flush with DNA polymerase I (Klenow), mid DNA from several amplicillin resistant transformants further digested with BamHI, treated with BAP and electro were selected for analysis, and one plasmid DNA demon phoresed on a 6 percent polyacrylamide gel. The large vector strated the proper construction, and was designated pKCE fragment (fragment F) was isolated and purified. Atrp207-I*. 50 In a three fragment ligation, 50 ng fragment D, 100 ng E.1.8 Construction of a Plasmid Vector for Direct Expres fragment E, and 100 ng fragment F were ligated overnight at sion of Mouse Mature Anti-CEA Heavy (Gamma 1) Chain 4° in a 20 Jul reaction mixture and used to transform E. coli Gene, pyCEAtrp207-1* K12 strain 294. The plasmid DNAs from 12 amplicillin resis FIG. 7 illustrates the construction of pyCEAtrp207-1*. tant transformants were analyzed for the correct construction This plasmid was constructed in two parts beginning with 55 and the nucleotide sequence Surrounding the initiation codon construction of the C-terminal region of the gamma 1 gene. was verified to be correct for the plasmid named pyCEAInt2. 5ug of plasmid pHGH207-1 was digested with Ava I, The expression plasmid, pyCEAtrp207-I used for expres extended to blunt ends with DNA polymerase I large frag sion of the heavy chain gene is prepared by a 3-way ligation ment (Klenow fragment), extracted with phenol/CHCl, and using the large vector fragment from pBR322(XAP) (supra) ethanol precipitated. The DNA was digested with BamH I 60 and two fragments prepared from pyCEAInt2. treated with BAP and the large fragment (fragment A) was pBR322(XAP) was treated as above by digestion with purified by 6 percent polyacrylamide gel electrophoresis and EcoR1, blunt ending with DNA polymerase (Klenow) in the electroelution. presence of dNTPs, followed by digestion with Pst I, and ~5ug of py11 was digested with Pst I and the gamma chain isolation of the large vector fragment by gel electrophoresis. cDNA insert fragment containing the C-terminal portion of 65 A 1543 base pair fragment from pyCEAInt2 containing trp the gene was purified, digested with Ava II followed by exten promoter linked with the N-terminal coding region of the sion of the Ava II cohesive ends with Klenow, followed by Taq heavy chain gene was isolated by treating pyCEAInt2 with Pst US 7,923,221 B1 23 24 I followed by BamHI, and isolation of the desired fragment FIG.8C shows results developed by Western blot from four using PAGE. The 869 base pair fragment containing the colonies of double transformed cells 24 hours after IAA addi C-terminal coding portion of the gene was prepared by partial tion (lanes 4-7). Lanes 1-3 are varying amounts of mono digestion of pyCEAInt2 with Ava I, blunt ending with Kle clonal gamma chain controls, lanes 8 and 9 are untransformed now, and subsequent digestion with BamH I, followed by and pyCEAtrp207-1 transformed cell extracts, respectively. purification of the desired fragment by gel electrophoresis. In another quantitative assay, frozen, transformed E. coli The aforementioned three fragments were then ligated cells grown according to E.1.10 (below) were lysed by heat under standard conditions using T4 DNA ligase, and a liga ing in sodium dodecyl sulfate (SDS)/y-mercaptoethanol cell tion mixture used to transform E. coli strain 294. Plasmid lysis buffer at 100°. Aliquots were loaded on an SDS poly DNAs from several tetracycline resistant transformants were 10 acrylamide gel next to lanes loaded with various amounts of analyzed; one plasmid DNA demonstrated the proper con hybridoma anti-CEA. The gel was developed by the Western struction and was designated pyCEAtrp207-1*. blot, Burnett (supra), using 'I-labeled sheep anti-mouse E.1.9 Production of Immunoglobulin Chains by E. coli IgG antibody from New England Nuclear. The results are E. coli strain W3110 (ATTC No. 27325) was transformed shown in FIG. 9. The figure shows that the E. coli products with pyCEAtrp207-1* or pKCEAtrp207-1* using standard 15 co-migrate with the authentic hybridoma chains, indicating techniques. no detectable proteolytic degradation in E. coli. Heavy chain To obtain double transformants, E. coli strain W3110 cells from mammalian cells is expected to be slightly heavier than were transformed with a modified pKCEAtrp207-1, pKCE E. colimaterial due to glycosylation in the former. Using the Atrp207-1A, which had been modified by cleaving a Pst hybridoma lanes as a standard, the following estimates of I-Pvu I fragment from the amp' gene and religating. Cells heavy and light chain production were made: transformed with pKCEAtrp207-1A are thus sensitive to ampicillin but still resistant to tetracycline. Successful trans formants were retransformed using pyCEAInt2 which con fers resistance to amplicillin but not tetracycline. Cells con (Per gram of cells) taining both pKCEAtrp207-1“A and pyCEAInt2 thus 25 E. coli (W3110/pyCE Atrp207-1*) 5 mg Y identified by growth in a medium containing both amplicillin E. coli (W3110/pKCEAtrp207-1*) 1.5 mg K and tetracycline. E. coli (W3110/pKCE Atrp207-1*A, pyCEAInt2) 0.5 mg K, 1.0 mgy To confirm the production of heavy and/or light chains in the transformed cells, the cell samples were inoculated into E.1.10 Reconstitution of Antibody from Recombinant K M9 tryptophan free medium containing 10 ug/ml tetracy 30 and Gamma Chains cline, and induced with indoleacrylic acid (IAA) when the In order to obtain heavy and light chain preparations for OD 550 reads 0.5. The induced cells were grown at 37° C. reconstitution, transformed cells were grown in larger during various time periods and then spun down, and Sus batches, harvested and frozen. Conditions of growth of the pended in TE buffer containing 2 percent SDS and 0.1 M variously transformed cells were as follows: B-mercaptoethanol and boiled for 5 minutes. A 10x volume of 35 E. coli (W3110/pyCEAtrp207-1*) were inoculated into acetone was added and the cells kept at 22°C. for 10 minutes, 500 ml LB medium containing 5 g/ml tetracycline and then centrifuged at 12,000 rpm. The precipitate was sus grown on a rotary shaker for 8 hours. The culture was then pended in O’Farrell SDS sample buffer (O'Farrell, P. H., J. transferred to 10 liters of fermentation medium containing Biol. Chem., 250: 4007 (1975)); boiled 3 minutes, recentri yeast nutrients, salts, glucose, and 2 Lig/ml tetracycline. Addi fuged, and fractionated using SDS PAGE (10 percent), and 40 tional glucose was added during growth and at OD 550-20, stained with silver stain (Goldman, D. et al., Science 211: indoleacrylic (IAA), a trp derepressor, was added to a con 1437 (1981)); or subjected to Western blot using rabbit anti centration of 50 ug/ml. The cells were fed additional glucose mouse IgG (Burnett, W. N., et al., Anal. Biochem. 112: 195 to a final OD 550–40, achieved approximately 6 hours from (1981)), for identification light chain and heavy chain. the IAA addition. Cells transformed with pyCEAtrp207-1* showed bands 45 E. coli (W3110) cells transformed with pKCEA trp 207-1* upon SDS PAGE corresponding to heavy chain molecular and double transformed (with pKCEAtrp207-1* A and weight as developed by silver stain. Cells transformed with pyCEAInt2) were grown in a manner analogous to that pKCEAtrp207-1 showed the proper molecular weight band described above except that the OD 550 six hours after IAA for light chain as identified by Western blot; double trans addition at harvest was 25-30. formed cells showed bands for both heavy and light chain 50 The cells were then harvested by centrifugation, and fro molecular weight proteins when developed using rabbit anti ZC. mouse IgG by Western blot. These results are shown in FIGS. E.2 Assay Method for Reconstituted Antibody 8A, 8B, and 8C. Anti-CEA activity was determined by ELISA as a criterion FIG. 8A shows results developed by silver stain from cells for successful reconstitution. Wells of microtiter plates (Dy transformed with pyCEAtrp207-1. Lane 1 is monoclonal 55 natech Immulon) were saturated with CEA by incubating 100 anti-CEA heavy chain (standard) from CEA.66-E3. Lanes ul of 2-5 ug CEA/ml solution in 0.1M carbonate buffer, pH 2b-5b are timed samples 2 hrs, 4 hrs, 6 hrs, and 24 hrs after 9.3 for 12 hours at room temperature. The wells were then IAA addition. Lanes 2a-5a are corresponding untransformed washed 4 times with phosphate buffered saline (PBS), and controls.; Lanes 2c-5c are corresponding uninduced transfor then saturated with BSA by incubating 200 ul of 0.5 percent mants. 60 BSA in PBS for 2 hours at 37° C., followed by washing 4 FIG. 8B shows results developed by Western blot from times with PBS. Fifty microliters of each sample was applied cells transformed with pKCEAtrp207-1. Lanes 1b-6b are to each well. A standard curve (shown in FIG. 10), was run, extracts from induced cells immediately, 1 hr, 3.5 hrs, 5 hrs, 8 which consisted of 50 ul samples of 101g, 5ug, 1 Jug, 500 ng, hrs, and 24 hrs after IAA addition, and 1a-6a corresponding 100 ng, 50 ng, 10ng, 5 ng and 1 nganti-CEA/ml in 0.5 percent uninduced controls. Lane 7 is an extract from a pyCE 65 BSA in PBS, plus 50 ul of 0.5 percent BSA in PBS alone as a Atrp207-1° control, lanes 8,9, and 10 are varying amounts of blank. All of the samples were incubated in the plate for 90 anti CEA-kappa chain from CEA.66-E3 cells. minutes at 37° C. US 7,923,221 B1 25 The plates were then washed 4 times with PBS, and sheep -continued anti-mouse IgG-alkaline phosphate (TAGO, Inc.) was Percent applied to each well by adding 100 ul of an enzyme concen ng/ml (CO tration of 24 units/ml in 0.5 percent BSA in PBS. The solution anti-CEA bination was incubated at 37° C. for 90 minutes. The plates were E. coli (W3110/pKCEAtrp207-1*), plus Y-SS0, 848 O.33 washed 4 times with PBS before adding the substrate, 100 ul E. coli (W3110/pKCEAtrp207-1*A, pyCEAInt2) 1580 O.76 of a 0.4 mg/ml solution of p-nitrophenylphosphate (Sigma) in Hybridoma anti-CEA K-SSO and Y-SSOs S4O O.40 ethanolamine buffered saline, pH 9.5. The substrate was incu bated 90 minutes at 37°C. for color development. The Also of each well was read by the Microelisa Auto 10 E.4 Preparation of Chimeric Antibody Reader (Dynatech) set to a threshold of 1.5, calibration of 1.0 FIGS. 11 and 12 show the construction of an expression and the 0.5 percent BSA in PBS (Blank) well set to 0.000. The vector for a chimeric heavy (gamma) chain which comprises Also data was tabulated in RS-1 on the VAX system, and the the murine anti CEA variable region and human Y-2 constant standard curve data fitted to a four-parameter logistic model. region. The unknown samples’ concentrations were calculated based 15 A DNA sequence encoding the human gamma-2 heavy on the Aso data. chain is prepared as follows: the cDNA library obtained by E.3 Reconstitution of Recombinant Antibody and Assay standard techniques from a human multiple myeloma cell line Frozen cells prepared as described in paragraph E.1.10 is probed with 5' GGGCACTCGACACAA 3' to obtain the were thawed in cold lysis buffer 10 mM Tris HCl, pH 7.5, 1 plasmid containing the cDNA insert for human gamma-2 mM EDTA, 0.1M NaCl, 1 mM phenylmethylsulfonyl fluo chain (Takahashi, et al., Cell, 29: 671 (1982), incorporated ride (PMSF) and lysed by sonication. The lysate was par herein by reference), and analyzed to verify its identity with tially clarified by centrifugation for 20 mins at 3,000 rpm. The the known sequence in human gamma-2 (Ellison, J., et al., Supernatant was protected from proteolytic enzymes by an Proc. Natl. Acad. Sci. (USA), 79: 1984 (1982) incorporated additional 1 mM PMSF, and used immediately or stored herein by reference). frozen at -80°C.; frozen lysates were neverthawed more than 25 As shown in FIG. 11, two fragments are obtained from this OCC. cloned human gamma 2 plasmid (py2). The first fragment is The S-sulfonate of E. coli produced anti-CEA heavy chain formed by digestion with PvulI followed by digestion with (Y) was prepared as follows: Recombinant E. coli cells trans Ava III, and purification of the smaller DNA fragment, which formed with pyCEAtrp207-1, which contained heavy chain contains a portion of the constant region, using 6 percent as insoluble bodies, were lysed and centrifuged as above; the 30 PAGE. The second fragment is obtained by digesting the py2 pellet was resuspended in the same buffer, Sonicated and with any restriction enzyme which cleaves in the 3' untrans re-centrifuged. This pellet was washed once with buffer, then lated region of Y2, as deduced from the nucleotide sequence, suspended in 6Mguanidine HCl, 0.1M Tris HCl, pH 8, 1 mM filling in with Klenow and dNTPs, cleaving with Ava III, and EDTA, 20 mg/ml sodium sulfite and 10 mg/ml sodium isolating the smaller fragment using 6 percent PAGE. (The tetrathionate and allowed to react at 25° for about 16 hrs. The 35 choice of a two step, two fragment composition to Supply the reaction mixture was dialyzed against 8M urea, 0.1M Tris PvulI-3' untranslated fragment provides a cleaner path to HCl, pH 8, and stored at 4°, to give a 3 mg/ml solution of product due to the proximity of the AvalII site to the 3 termi Y-SSO. nal end thus avoiding additional restriction sites in the gene 650 ul of cell lysate from cells of various E. coli strains sequence matching the 3' untranslated region site.) producing various IgG chains, was added to 500 mg urea. To 40 pyCEA207-1* is digested with EcoR1, treated with Klenow this was added B-mercaptoethanol to 20 mM, Tris-HCl, pH and dNTPs to fill in the cohesive end, and digested with Pvu 8.5 to 50 mM and EDTA to 1 mM, and in some experiments, II, the large vector fragment containing promoter isolated by Y-SSO was added to 0.1 mg/ml. After standing at 25° for 6 percent PAGE. 30-90 mins., the reaction mixtures were dialyzed at 4 against The location and DNA sequence surrounding the PvulI site a buffer composed of 0.1M sodium glycinate, pH 10.8, 0.5M 45 in the mouse gamma-1 gene are identical to the location and urea, 10 mM glycine ethyl ester, 5 mM reduced glutathione, DNA sequence surrounding the PvulI site in the human 0.1 mM oxidized glutathione. This buffer was prepared from gamma-2 gene. N-saturated water and the dialysis was performed in a The plasmid resulting from a three way ligation of the capped Wheaton bottle. After 16-48 hours, dialysis bags were foregoing fragments, pChiml, contains, under the influence transferred to 4 phosphate buffered saline containing 1 mM 50 of trp promoter, the variable and part of the constant region of PMSF and dialysis continued another 16-24 hrs. Dialysates murine anti-CEA gamma 1 chain, and a portion of the gamma were assayed by ELISA as described in paragraph E.2 for 2 human chain. pChim1 will, in fact, express achimeric heavy ability to bind CEA. The results below show the values chain when transformed into E. coli, but one wherein the obtained by comparison with the standard curve inxing/ml change from mouse to human does not take place at the anti-CEA. Also shown are the reconstitution efficiencies cal 55 variable to constant junction. culated from the ELISA responses, minus the background FIG. 12 shows modification of pChim1 to construct (108 ng/ml) of cells producing K chain only, and from esti pChim2 so that the resulting protein from expression will contain variable region from murine anti CEA antibody and mates of the levels of Y and K chains in the reaction mixtures. constant region from the human Y-2 chain. First, a fragment is 60 prepared from pChim1 by treating with Nco I, blunt ending with Klenow and dNTPs, cleaving with Pvu II, and isolating Percent the large vector fragment which is almost the complete plas ng ml (CO mid except for short segment in the constant coding region for anti-CEA bination mouse anti CEA. A second fragment is prepared from the E. coli W3110 producing IFN-CA (control) O 65 previously described py2 by treating with Pvu II, followed by E. coli (W3110/pKCEAtrp207-1*) 108 treating with any restriction enzyme which cleaves in the variable region, blunt ending with Klenow and dNTPs and US 7,923,221 B1 27 28 isolating the short fragment which comprises the junction Expression of the anti-CEA gamma 1 chain Fab fragment between variable and constant regions of this chain. rather than complete heavy chain requires that a termination Ligation of the foregoing two fragments produces an inter codon be constructed at the appropriate location in the gene. mediate plasmid which is correct except for an extraneous For this, the 260 bpNco I-Nde I DNA fragment from 20 g of DNA fragment which contains a small portion of the constant 5 the py298 was isolated and purified. A 13 nucleotide DNA region of the murine anti CEA antigen, and a small portion of primer, the complement of which encodes the last 3 C-termi the variable region of the human gamma chain. This repair nal amino acids of the Fab gene and 2 bases of the 3 needed for can be made by excising the Xba I to Pvu II fragment and the stop codon, was synthesized by the phosphotriester cloning into M13 phage as described by Messing et al., method (supra). The probe hybridizes to nucleotides 754 to Nucleic Acids Res. 9:309 (1981), followed by in vitro site 10 directed deletion mutagenesis as described by Adelman, et 767 (FIG. 4) which has the following sequence: al., DNA, in press (1983) which is incorporated herein by reference. The Xba I-Pvu II fragment thus modified is ligated AspCysGly.Stop back into the intermediate plasmid to form pChim2. This s' GGGATTGTGGTTG 3." plasmid then is capable of expressing in a suitable host a 15 cleanly constructed murine variable/human constant chi The third base of the stop codon is provided by the terminal meric heavy chain. nucleotide of the filled-in HindIII site from pBR322 cleavage In an analogous fashion, but using mRNA templates for described above. 500 ng of this primer was used in a primer cDNA construction for human kapparather than Y chain, the repair reaction by phosphorylation at the 5' end in a reaction expression plasmid for chimeric light chain is prepared. 2O with 10 units T4DNA kinase containing 0.5 mMATP in 20 ul, The foregoing two plasmids are then double transformed and mixing with ~200 ng of the Nco I-Nde I DNA fragment. into E. coli W31 10, the cells grown and the chains reconsti The mixture was heat denatured for 3 minutes at 95° and tuted as set forth in paragraph E.1-E.3 Supra; quenched in dry-ice ethanol. The denatured DNA solution E.5 Preparation of Altered Murine Anti-CEA Antibody was made 60 mM. NaCl, 7 mM MgCl2, 7 mM Tris HCl (pH E.5.1 Construction of Plasmid Vectors for Direct Expres- 25 7.4), 12 mM in each dNTP and 12 units DNA Polymerase sion of Altered Murine Anti-CEA Heavy Chain Gene I-Large Fragment was added. After 2 hours incubation at 37° The cysteine residues, and the resultant disulfide bonds in C., this primer repair reaction was phenol/CHCl extracted, the region of amino acids 216-230 in the constant region of ethanol precipitated, digested with BamHI and the reaction murine anti-CEA heavy chain are Suspected to be important electrophoresed through a 6 percent polyacrylamide gel. ~50 for complement fixation (Klein, et al., Proc. Natl. Acad. Sci., 30 ng of the 181 bp blunt end to BamHI DNA fragment, frag (USA), 78: 524 (1981)) but not for the antigen binding prop ment III, was isolated and purified. erty of the resulting antibody. To decrease the probability of ~100 ng of fragment I, -100 ng each of fragments II and III incorrect disulfide bond formation during reconstitution were ligated overnight and transformed into E. coli K12 strain according to the process of the invention herein, the nucle 294. Plasmid DNA from several tetracycline resistant trans otides encoding the amino acid residues 226-232 which 35 formants was analyzed for the proper construction and the includes codons for three cysteines, are deleted as follows: nucleotide sequence through the repair blunt end filled-in A “deleter” deoxyoligonucleotide, 5' CTAACACCATGT HindIII junction was determined for verification of the TGA CAGGGT is used to delete the relevant portions of the gene stop codon. from pyCEAtrp207-1 by the procedure of Wallace, et al., E.6.2 Production of Fab Protein Science, 209: 1396 (1980) or of Adelman, et al., DNA (in 40 The plasmid prepared in E.6.1 is transformed into an E. coli press) 1983. Briefly, the “deleter deoxyoligonucleotide is strain previously transformed with pKCEAtrp207-1* as annealed with denatured pyCEAtrp207-1 DNA, and primer described above. The cells are grown, extracted for recombi repair synthesis carried out in vitro, followed by Screening by nant antibody chains and the Fab protein reconstituted as hybridization of presumptive deletion clones with p described in E.1.10. labelled deleter sequence. 45 E.5.2 Production of Cysteine Deficient Altered Antibody The invention claimed is: The plasmid prepared in E.5.1 is transformed into an E. coli 1. A method for making an antibody heavy chain or frag strain previously transformed with pKCEAtrp207-1* as ment thereof and an antibody light chain or fragment thereof described above. The cells are grown, extracted for recombi each having specificity for a desired antigen, wherein the nant antibody chains, and the altered antibody reconstituted 50 heavy chain or fragment thereof comprises a human constant as described in E.1.10. region sequence and a variable region comprising nonhuman E.6 Preparation of Fab mammalian variable region sequences, the method compris E.6.1 Construction of a Plasmid Vector for Direct Expres ing culturing a recombinant host cell comprising DNA encod sion of Murine Anti-CEA Gamma 1 Fab Fragment Gene ing the heavy chain or fragment thereof and the light chain or pyCEAFabtrp207-1* 55 fragment thereof and recovering the heavy chain or fragment FIG. 13 presents the construction of pyCEAFabtrp207-1*. thereof and light chain or fragment thereof from the host cell 5ug of pBR322 was digested with HindIII, the cohesive ends culture. made flush by treating with Klenow and dNTPs; digested 2. The method of claim 1 wherein the light chain or frag with Pst I, and treated with BAP. The large vector fragment, ment thereof comprises a human constant region sequence fragment I, was recovered using 6 percent PAGE followed by 60 and a variable region comprising non human mammalian electroelution. variable region sequences. 5ug of pyCEAtrp207-1 was digested with both BamH I 3. The method of claim 1 wherein the host cell comprises a and Pst I and the ~1570 bp DNA fragment (fragment II) vector comprising DNA encoding the heavy chain or frag containing the trp promoter and the gene sequence encoding ment thereof and DNA encoding the light chain or fragment the variable region continuing into constant region and fur- 65 thereof. ther into the anti-CEA gamma 1 chain hinge region, was 4. The method of claim 1 wherein the host cell comprises a isolated and purified after electrophoresis. vector comprising DNA encoding the heavy chain or frag US 7,923,221 B1 29 30 ment thereof and a further vector comprising DNA encoding ing a human constant region sequence and a variable region the light chain or fragment thereof. comprising non human mammalian variable region 5. The method of claim 1 wherein the nonhuman mamma Sequences. lian variable region sequences are murine. 23. A recombinant host cell comprising the vector of claim 6. The method of claim 1 wherein the host cell is a prokary 22. otic cell. 24. A recombinant host cell comprising (a) a vector com 7. The method of claim 6 wherein the prokaryotic cell is an prising DNA encoding an antibody heavy chain or fragment E. coli cell. thereof comprising a human constant region sequence and a 8. The method of claim 1 wherein the host cell is an eukary variable region comprising non human mammalian variable 10 region sequences and (b) a vector comprising DNA encoding otic cell. an antibody light chain or fragment thereof comprising a 9. The method of claim 8 wherein the eukaryotic cell is a human constant region sequence and a variable region com mammalian cell. prising nonhuman mammalian variable region sequences. 10. The method of claim 9 wherein the mammalian cell is 25. A method for making an antibody heavy chain or frag selected from the group consisting of a VERO, HeLa, Chinese 15 ment thereof and an antibody light chain or fragment thereof Hamster Ovary (CHO), W138, BHK, COS-7 and MDCK each having specificity for a desired antigen, wherein the cell. heavy chain or fragment thereof comprises a variable region 11. The method of claim 10 wherein the mammalian cell is sequence and a human constant region sequence, the method a CHO cell. comprising culturing a recombinant host cell comprising 12. The method of claim 10 wherein the mammalian cell is DNA encoding the heavy chain or fragment thereof and the a COS-7 cell. light chain or fragment thereof and recovering the heavy 13. The method of claim 8 wherein the eukaryotic cell is a chain or fragment thereof and light chain or fragment thereof yeast cell. from the host cell culture. 14. The method of claim 13 wherein the yeast cell is a 26. The method of claim 25 wherein the light chain or Saccharomyces cerevisiae cell. 25 fragment thereof comprises a variable region sequence and a 15. A method for making an antibody or antibody fragment human constant region sequence. capable of specifically binding a desired antigen, wherein the 27. The method of claim 25 wherein the host cell comprises antibody or antibody fragment comprises (a) an antibody a vector comprising DNA encoding the heavy chain or frag heavy chain or fragment thereof comprising a human con ment thereof and DNA encoding the light chain or fragment stant region sequence and a variable region comprising non 30 thereof. 28. The method of claim 25 wherein the host cell comprises human mammalian variable region sequences and (b) an anti a vector comprising DNA encoding the heavy chain or frag body light chain or fragment thereof comprising a human ment thereof and a further vector comprising DNA encoding constant region sequence and a variable region comprising the light chain or fragment thereof. non human mammalian variable region sequences, the 35 29. The method of claim 25 wherein the host cell is a method comprising coexpressing the heavy chain or fragment prokaryotic cell. thereof and the light chain or fragment thereof in a recombi 30. The method of claim 29 wherein the prokaryotic cell is nant host cell. an E. coli cell. 16. The method of claim 15 further comprising recovering 31. The method of claim 25 wherein the host cell is a the antibody or antibody fragment from a cell culture com 40 eukaryotic cell. prising the recombinant cell. 32. The method of claim 31 wherein the eukaryotic cell is 17. The method of claim 15 which results in the production a mammalian cell. of an antibody fragment. 33. The method of claim 32 wherein the mammalian cell is 18. The method of claim 17 wherein the antibody fragment selected from the group consisting of a VERO, HeLa, Chinese is an F(ab')2 fragment. 45 Hamster Ovary (CHO), W 138, BHK, COS-7 and MDCK 19. The method of claim 17 wherein the antibody fragment cell. is a Fab fragment. 34. The method of claim 32 wherein the mammalian cell is 20. The method of claim 15 which results in the production a CHO cell. of an antibody. 35. The method of claim 32 wherein the mammalian cell is 21. A method for making an antibody capable of specifi 50 a COS-7 cell. cally binding a desired antigen, the antibody comprising 36. The method of claim 31 wherein the eukaryotic cell is heavy and light immunoglobulin polypeptide chains each a yeast cell. comprising a human constant region sequence and a variable 37. The method of claim 36 wherein the yeast cell is a region comprising non human mammalian variable region Saccharomyces cerevisiae cell. sequences, the method comprising the steps of (a) transform 55 38. A method for making an antibody or antibody fragment ing a recombinant host cell with a replicable expression vec capable of specifically binding a desired antigen, wherein the tor comprising DNA encoding the heavy immunoglobulin antibody or antibody fragment comprises (a) an antibody polypeptide chain and a replicable expression vector com heavy chain or fragment thereof comprising a variable region prising DNA encoding the light immunoglobulin polypeptide sequence and a human constant region sequence and (b) an chain, wherein each of the DNAs is operably linked to a 60 antibody light chain or fragment thereof comprising a vari promoter, and (b) culturing the host cell to produce a host cell able region sequence and a human constant region sequence, culture that expresses said antibody. the method comprising coexpressing the heavy chain or frag 22. A replicable expression vector comprising DNA ment thereof and the light chain or fragment thereof in a encoding an antibody heavy chain or fragment thereof and an recombinant host cell. antibody light chain or fragment thereof each having speci 65 39. The method of claim 38 further comprising recovering ficity for a desired antigen, the heavy chain or fragment the antibody or antibody fragment from a cell culture com thereof and the light chain or fragment thereof each compris prising the recombinant cell. US 7,923,221 B1 31 32 40. The method of claim 38 which results in the production 45. A replicable expression vector comprising DNA of an antibody fragment. encoding an antibody heavy chain or fragment thereof and an 41. The method of claim 38 wherein the antibody fragment antibody light chain or fragment thereof each having speci is an F(ab) fragment. ficity for a desired antigen, the heavy chain or fragment 42. The method of claim 40 wherein the antibody fragment thereof and the light chain or fragment thereof each compris is a Fab fragment. ing a variable region sequence and a human constant region 43. The method of claim 38 which results in the production of an antibody. Sequence. 44. A method for making an antibody capable of specifi 46. A recombinant host cell comprising the vector of claim cally binding a desired antigen, the antibody comprising 45. heavy and light immunoglobulin polypeptide chains each 10 47. A recombinant host cell comprising (a) a vector com comprising a variable region sequence and a human constant prising DNA encoding an antibody heavy chain or fragment region sequence, the method comprising the steps of (a) trans thereof comprising a variable region sequence and human forming a recombinant host cell with a replicable expression constant region sequence and (b) a vector comprising DNA vector comprising DNA encoding the heavy immunoglobulin encoding an antibody light chain or fragment thereof com polypeptide chain and a replicable expression vector com 15 prising a variable region sequence and a human constant prising DNA encoding the light immunoglobulin polypeptide region sequence. chain, wherein each of the DNAs is operably linked to a promoter, and (b) culturing the host cell to produce a host cell culture that expresses said antibody.